Bifunctional antibiotics for targeting rRNA and resistance-causing enzymes and for inhibition of anthrax lethal factor

ABSTRACT

A novel group of aminoglycosides which share some structural features of currently available aminoglycosides with regard to the backbone, while also having significant structural differences, is disclosed. The similarity enables these aminoglycosides to be highly potent and effective antibiotics, while the significant differences enable these aminoglycosides to reduce or even block antibiotic resistance. The aminoglycosides of the present invention are suitable for inhibition of antrax lethal factor, hence are suitable for use as a cure for anthrax.

This application is a continuation-in-part of PCT Patent Application No.PCT/IL2004/000490, filed Jun. 9, 2004, which claims priority from U.S.Pat. application Ser. No. 10/829,976, filed Apr. 23, 2004, and U.S.Provisional Patent Applications Nos. 60/540,359, filed Feb. 2, 2004, and60/484,293, filed Jul. 3, 2003. This application also claims the benefitof priority from U.S. Provisional Patent Application No. 60/608,372,filed Sep. 10, 2004. The teachings of the above applications are herebyincorporated by reference as if fully set forth herein.

FIELD OF THE INVENTION

The present invention relates to bi-functional antibiotics, and inparticular to aminoglycosides which are capable of reducing the efficacyof and/or blocking antibiotic resistance. The aminoglycosides of thepresent invention are suitable for inhibition of anthrax lethal factor,hence are suitable for use as a cure for anthrax.

BACKGROUND OF THE INVENTION

The rapid spread of antibiotic resistance in pathogenic bacteria hasprompted a continuing search for new agents capable of antibacterialactivity. Indeed, microbiologists today warn of a “medical disaster”which could lead back to the era before penicillin, when even seeminglysmall infections were potentially lethal. Thus, research into the designof new antibiotics is of high priority (1-3). One way to delay theemergence of antibiotic-resistance is to develop new synthetic materialsthat can selectively inhibit bacterial enzymes, via novel mechanisms ofaction. However, this approach is both time-consuming and financiallyprohibitive, yet remains indispensable if an acceptable level of care isto be provided in the immediate future. On the other hand, it may beless costly in time and money to employ strategies to circumventexisting bacterial resistance mechanisms and thereby to restoreusefulness to antibacterials that have become compromised by resistance(4). The remarkable advances in recent years in elucidating themechanisms of resistance to various clinical antibiotics on themolecular level provide complementary tools to this approach viastructure-based and mechanism-based design.

One example of an important group of antibiotics which could benefitfrom such a redesign is the aminoglycoside class of antibiotics.Aminoglycosides (as shown in prior art FIG. 1) are highly potent,broad-spectrum antibiotics with many desirable properties for thetreatment of life-threatening infections (5). Their history begins in1944 with streptomycin and was thereafter marked by the successiveintroduction of a series of milestone compounds (neomycin, kanamycin,gentamycin, tobramycin, and others), which definitively established theusefulness of this class of antibiotics for the treatment ofgram-negative bacillary infections (6). It is believed thataminoglycosides exert their therapeutic effect by interfering withtranslational fidelity during protein synthesis via interaction with theA-site rRNA on the 16S domain of the ribosome (7,8). Recent achievementsin ribosome structure determination have provided fascinating newinsights into the decoding site of the ribosome at high resolution andhow aminoglycosides might induce misreading of the genetic code.

Unfortunately, prolonged clinical use of currently availableaminoglycosides has resulted in effective selection of resistance tothis family of antibacterial agents (9). Presently, resistance to theseagents is widespread among pathogens worldwide which severely limitstheir usefulness. The primary mechanism for resistance toaminoglycosides is the bacterial acquisition of enzymes which modifythis family of antibiotics by acetyltransferase (AAC), adenyltransferase(ANT), and phosphotransferase (APH) activities (as shown in prior artFIG. 2). Among these enzyme families, aminoglycoside3′-phosphotransferases [APH(3′)s], of which seven isozymes are known,are widely represented. These enzymes catalyze transfer of γ-phosphorylgroup of ATP to the 3′-hydroxyl of many aminoglycosides, rendering theminactive because the resulted phosphorylated antibiotics no longer bindto the bacterial ribosome with high affinity. Due to the unusually broadspectrum of aminoglycosides that can be detoxified by APH(3′) enzymes,much effort has been put into understanding the structural basis fortheir promiscuity in substrate recognition and catalysis (10).

To tackle the problem of antibiotic resistance, many structural analogsof natural aminoglycosides have been synthesized over the past decade(11). In the majority of these studies a minimal structural motif, whichis common for a series of structurally related aminoglycosides, has beenidentified and used as a scaffold for the construction of diverseanalogs as potential new antibiotics (12). Some of the designedstructures showed considerable antibacterial activities. Since thestructural and mechanistic information on the target(s) ofaminoglycosides and their respective resistance enzymes has only beganto emerge in the past few years, this information stimulated noveldevelopments in the de novo design of molecules that bind to theribosomal target site and simultaneously are poor substrates forresistance-causing enzymes (13, 14). These results and design principleshold the promise of the generation of a large series of designerantibiotics uncompromised by the existing mechanisms of resistance.

In view of recent events, one particular disease for which effectivetherapeutic strategies are urgently required is anthrax.

Anthrax is an infectious disease caused by toxigenic strains of theGram-positive Bacillus anthracis (15). If inhaled, B. anthracis sporesrapidly reach the regional lymphonodes of the lungs where they germinateand release anthrax toxins (16). These toxins inhibit the adaptiveimmune response, thereby enabling the bacteria to reach the blood systemwhere they cause bacteraemia and toxaemia, which rapidly kills the host.Non-toxigenic strains of B. anthracis are poorly pathogenic indicatingthat the anthrax toxins play a major role from the very beginning ofinfection to death. Since anthrax is asymptomatic until the bacteriumreach the blood (15, 16), the development of anti-toxin therapeutics forpreventive use or in combination with antibiotics, is of high urgency(17). Alternatively, and even preferably, the development ofbifunctional substances that would inactivate the released toxins and inaddition would function as an antibiotic would be highly beneficial.

The anthrax toxins consist of three proteins: protective antigen (PA),edema factor (EF), and lethal factor (LF) (18). Being individuallynontoxic, their toxic effects during anthrax infection requirecooperation: PA binds to a cell surface receptor and forms an oligomericpore that translocates both EF and LF into the cytosol of target cells.Once inside the cell, EF causes edema via Ca²⁺/calmodulin-dependentadenylate cyclase activity. LF is a zinc-dependent endopeptidase thatspecifically cleaves most isoforms of mitogen-activated protein kinasekinases, thereby inhibiting one or more signaling pathways of the hostmacrophage (19). Through a mechanism that is not yet well understood,this results in the death of the host. Strains of B. anthracis deficientin EF remain pathogenic, while those lacking LF become attenuated. LF istherefore considered the dominant virulence factor of anthrax (20).Consequently, an intensive search for specific inhibitors of LF has beenperformed during the last years (17, 21-22).

The prior art does not teach or suggest a highly effective group ofaminoglycosides which both share certain structural features ofcurrently available aminoglycosides while also being able to reduce oreliminate antibiotic resistance. The prior art also does not teach orsuggest such aminoglycosides which have reduced side effects. The priorart also does not teach or suggest such aminoglycosides which arecapable of functioning both by inhibition of anthrax lethal factor, andas an antibiotic and are therefore highly effective for treatment ofanthrax.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, bifunctional antibiotics which both inactivatetoxins and function as an antibiotic, while reducing or eliminatingantibiotic resistance, and further resulting in reduced side effects.Such antibiotics would be highly beneficial in the treatment ofbacterial infections such as anthrax.

SUMMARY OF THE INVENTION

The present invention overcomes the deficiencies of the prior art byproviding a novel group of aminoglycosides which share some structuralfeatures of currently available aminoglycosides with regard to thebackbone, while also having significant structural differences. Thesimilarity enables these aminoglycosides to be highly potent andeffective antibiotics, while the significant differences enable theseaminoglycosides to reduce or even block antibiotic resistance. Theseaminoglycosides are highly effective for treatment of anthrax.

The present invention represents a new class of bifunctional antibioticsthat circumvent antibiotic resistance and that also have a highpotential for immediate therapeutic applications. Without wishing to belimited by a single hypothesis, it is believed that the aminoglycosidesof the present invention target both bacterial rRNA and inhibitresistance-causing enzymes. These aminoglycosides are therefore a newclass of semi-synthetic analogs of currently available aminoglycosides,which are useful for many different functions, including determiningimportant complementary information on their antibacterial activity,interaction with resistance-causing enzymes in the means of kinetics ofbinding and high-resolution three-dimensional structures of binary andternary complexes, and binding to the A-site rRNA. The new designstrategies, methodologies and principles developed and discussed in thepresent invention are also expected to be valuable for unravelingsimilar problems posed to other families of antibiotics and other drugs.

According to preferred embodiments of the present invention, compoundsbelonging to the novel group of aminoglycosides described above have thegeneral formula I:

wherein:

-   R₁ is a monosaccharide residue or an oligosaccharide residue;-   X and Y are independently oxygen or sulfur;-   R₂ and R₃ are each independently selected from the group consisting    of hydrogen, hydroxy, thiol, amine, alkyl, cycloalkyl, aryl, alkoxy,    aryloxy, thioalkoxy and thioaryloxy; and    wherein the carbon at the fifth position of ring B has an R    configuration or an S configuration;    and pharmaceutically acceptable salts thereof.

As used herein, the term “hydroxy” refers to a —OH group.

The term “thiol” refers to a —SH group.

The term “alkyl”, as used herein, refers to a saturated aliphatichydrocarbon including straight chain and branched chain groups.Preferably, the alkyl group has 1 to 20 carbon atoms. Whenever anumerical range; e.g., “1-20”, is stated herein, it implies that thegroup, in this case the alkyl group, may contain 1 carbon atom, 2 carbonatoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. Morepreferably, the alkyl is a medium size alkyl having 1 to 10 carbonatoms. Most preferably, unless otherwise indicated, the alkyl is a loweralkyl having 1 to 4 carbon atoms. The alkyl group may be substituted orunsubstituted. When substituted, the substituent group can be, forexample, halo, hydroxy, cyano, nitro, azo and amine, as these terms aredefined herein.

A “cycloalkyl” group refers to an all-carbon monocyclic or fused ring(i.e., rings which share an adjacent pair of carbon atoms) group whereinone of more of the rings does not have a completely conjugatedpi-electron system. Illustrative examples, without limitation, ofcycloalkyl groups are cyclopropane, cyclobutane, cyclopentane,cyclopentene, cyclohexane, cyclohexadiene, cycloheptane,cycloheptatriene, and adamantane. A cycloalkyl group may be substitutedor unsubstituted. When substituted, the substituent group can be, forexample, halo, hydroxy, cyano, nitro, azo and amine, as these terms aredefined herein.

An “aryl” group refers to an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. Examples,without limitation, of aryl groups are phenyl, naphthalenyl andanthracenyl. The aryl group may be substituted or unsubstituted. Whensubstituted, the substituent group can be, for example, halo, hydroxy,cyano, nitro, azo and amine, as these terms are defined herein.

An “alkoxy” group refers to both an —O-alkyl and an —O-cycloalkyl group,as defined herein.

An “aryloxy” group refers to both an —O-aryl group, as defined herein.

A “thioalkoxy” group refers to both an —S-alkyl group, and an—S-cycloalkyl group, as defined herein.

A “thioaryloxy” group refers to both an —S-aryl and an —S-heteroarylgroup, as defined herein.

An “azo” group refers to a —N═NR′ group, wherein R′ is hydrogen, alkyl,cycloalkyl or aryl.

A “halo” group refers to fluorine, chlorine, bromine or iodine.

An “amine” group refers to an —NR′R″ group where R′ is as definedhereinabove and R″ is as defined herein for R′.

A “nitro” group refers to a —NO₂ group.

A “cyano” group refers to a —C≡N group.

Preferably X is oxygen. Also preferably, Y is oxygen. Optionally andpreferably, R₁ is a monosaccharide residue. More preferably, themonosaccharide residue is a five-membered (furanose) or a six-membered(pyranose) monosaccharide residue. Also more preferably, themonosaccharide residue comprises at least one amine group and/or atleast one aminoalkyl group. Optionally and more preferably, the at leastone amine group and/or the at least one aminoalkyl group is at one ormore of positions 2, 3, 4 or 5. As used herein, the term “aminoalkyl”refers to an alkyl group, as defined hereinabove, which is substitutedby an amine group, as defined hereinabove. Optionally, at least oneaminoalkyl group is an aminomethyl group (—CH₂—NH₂).

Optionally and preferably, if the monosaccharide residue is a pyranosemonosaccharide residue, the aminomethyl group is at position 5.

Also optionally and preferably, if the monosaccharide residue is apyranose monosaccharide residue, the amine group is at one or more ofpositions 2, 3 or 4.

Also optionally and preferably, if the monosaccharide residue is afuranose monosaccharide residue, the aminoalkyl group is at position 4.

Optionally, the monosaccharide residue is a L-monosaccharide or aD-monosaccharide.

According to preferred embodiments of the present invention, R₁ is anoligosaccharide residue. Preferably, the oligosaccharide residuecomprises at least two monosaccharide residues, wherein each isindependently a five-membered (furanose) or a six-membered (pyranose)monosaccharide residue. More preferably, at least one of the at leasttwo monosaccharide residues comprises at least one amine group and/or atleast one aminoalkyl group. Most preferably, the at least one aminegroup is at position 2 of a pyranose monosaccharide residue. Also mostpreferably, the at least one aminoalkyl group is at position 5 of apyranose monosaccharide residue.

Optionally and preferably, the oligosaccharide comprises a furanosemonosaccharide linked to a pyranose monosaccharide.

Optionally, each of the at least two monosaccharide residues isindependently a D-monosaccharide or an L-monosaccharide.

Alternatively, the oligosaccharide residue comprises at least fourmonosaccharide residues, each being independently a five-membered(furanose) or a six-membered (pyranose) monosaccharide residue.Preferably, the oligosaccharide residue is a neomycin B residue. Otheroligosaccharide residues include, for example, a Paromomycin residue, aRibostamycin residue, a Gentamycin residue, a Amikacin residue, aNeamine residue, a Nebramine residue and a Tobramine residue.

According to other preferred embodiments of the present invention, X issulfur and R₁ is a monosaccharide residue. Preferably, themonosaccharide is a furanose monosaccharide residue.

According to still other preferred embodiments of the present invention,there are provided novel compounds each having the general formula II:

wherein:

-   Y is oxygen or sulfur;-   R₂ and R₃ are each independently hydrogen, hydroxy, thiol, amine,    alkyl, cycloalkyl, aryl, alkoxy, aryloxy, thioalkoxy and    thioaryloxy; and    wherein the carbon at the fifth position of ring B has an R    configuration or an S configuration;

and pharmaceutically acceptable salts thereof.

Preferably, Y is oxygen, and R₂ and R₃ are both hydroxy.

According to still other preferred embodiments of the present invention,there are provided novel compounds each having the general formula III:

wherein:

-   R₁ is a monosaccharide residue or an oligosaccharide residue;-   X is disulfide;-   R₂ and R₃ are each independently selected from the group consisting    of hydrogen, hydroxy, thiol, amine, alkyl, cycloalkyl, aryl, alkoxy,    aryloxy, thioalkoxy and thioaryloxy; and    wherein the carbon at the fifth position of ring B has an R    configuration or an S configuration;

and pharmaceutically acceptable salts thereof.

As used herein, the term “disulfide” refers to a compound having twosulfur atoms mutually bonded by a single bond.

Preferably, R₁ is an oligosaccharide residue and more preferably anoligosaccharide residue having at least four monosaccharide residues, asdescribed hereinabove.

Optionally and preferably, the oligosaccharide residue is a Neomycin Bresidue.

It should be noted that wherever reference is made to a general formulaor a specific compound according to the present invention,pharmaceutically acceptable salts are also optionally included.

All of the different structures of the preferred compounds according tothe present invention are shown in FIG. 16.

Without wishing to be limited by a single hypothesis, the presentinvention is believed to have better stability and greater resistance tobacterial enzymes for a number of reasons, including the optionalpresence of a thiol moiety at X, which is more resistant to hydrolysis.The presence of a monosaccharide or oligosaccharide at R₁ also increasesresistance to hydrolysis. Again without wishing to be limited by asingle hypothesis, resistance to hydrolysis is also believed to decreasetoxicity, as the compounds of the present invention are expected tohydrolyze within the body (outside of bacterial cells) at a lower rate,and hence to potentially produce fewer toxic degradation products.

Some background for the rational design of antibiotics is now provided.The first rationally designed semisynthetic aminoglycoside which wasselected for chemotherapeutic use is dibekacin (3′,4′-dideoxykanamycinB), developed in 1975 by Umezawa and co-workers (23a). The rationalebehind the development of this aminoglycoside variant was to overcomethe resistance to kanamycins due to bacterial enzymes that modify themby 3′-O-phosphorylation [APH(3′)]. Indeed dibekacin showed strongactivity not only against resistant staphylococci and Gram-negativebacteria, but also against Pseudomonas. This successful result boostedthe synthesis of numerous 3′-deoxy and 3′,4′-dideoxy derivatives ofother aminoglycosides, some of which were active against resistantbacteria producing APH(3′). Another approach to rationally designedsemi-synthetic aminoglycosides active against resistant bacteria is theacylation or alkylation of one or several amino groups ofaminoglycoside. This approach lead to the development of amikacin by1-N-acylation of kanamycin B with (S)-4-amino-2-hydroxybutiric acid(AHB), developed by Kawaguchi and co-workers and has been used in marketsince 1977 (23b). Similar approaches lead to the development ofnetilimicin (1985), isepamicin (1988), and arbekacin (1990), which aremarketed as chemotherapeutic agents, and were produced by 1—N-acylationwith different acylating groups (24). However, novel resistant bacteriaemerged to these antibiotics and again were shown to be dependent on newtypes of aminoglycoside-modifying enzymes.

To overcome the emerged resistance to amikacin, recently, Mobashery andco-workers (13) took advantage of the known 3D NMR structure forparomomycin bound to the A-site rRNA (25), and, by using dockingexperiments, a total of seven structures have been selected andsynthesized. AHB substitution at position N1 of designed molecules wasused, with the rationale that this group in amikacin is responsible forthe protection against a number of aminoglycoside-modifying enzymes thatcause N-acylation. Although two of these structures showed considerablyenhanced activity against different pathogenic and resistant strains ascompared to those of several conventional antibiotics, still theiractivities were mostly comparable to that of amikacin.

Most recently, Hanessian and co-workers (12g) used a similar approachand tried to mimic rings III and IV of paromomycin by attaching variousaminoalkyl substituents at C5 of tobramycin. For their design, they alsoemployed the available NMR and X-ray structural data of the complexes ofparomomycin and tobramycin with RNA sequences, as well as molecularmodeling. The 5-O-(2-guanidylethyl) ether of tobramycin was found to bethe most active analogue of this series, having similar antibacterialpotency to that of paromomycin.

During the last decade, more examples of synthetic variants of naturallyoccurring aminoglycosides have been reported (11). The strategies usedin the design of the majority of these mimetics were to start from apseudo-disaccharide (mostly neamine) as a minimum basic structure andincorporate therein various basic appendages at different positions. Thechoices of basic appendages, in the cases of rational design, relied onthe diversity of pKa, chain length, branching, and flexible topologies.In most cases however, the new analogs were either inactive or hadsignificantly lower activity than that of the parent structure, and onlya few new structures reported to date maintained an activity levelsimilar to that of the naturally occurring parent aminoglycosides. Thus,the construction of synthetic molecules providing better antibioticperformances than those of the naturally occurring drugs remains achallenging task.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

The term “comprising” means that other steps and ingredients that do notaffect the final result can be added. This term encompasses the terms“consisting of” and “consisting essentially of”.

The phrase “consisting essentially of” means that the composition ormethod may include additional ingredients and/or steps, but only if theadditional ingredients and/or steps do not materially alter the basicand novel characteristics of the claimed composition or method.

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures bypractitioners of the chemical, pharmacological, biological, biochemicaland medical arts.

An advantage of the compounds and/or the methods of the presentinvention is that bifunctional antibiotics are provided, which bothinactivate toxins and function as antibiotics.

Another object of the invention is to provide bifunctional antibioticswhich reduce or eliminate antibiotic resistance and which further resultin reduction of cytoxicity and other side-effects.

Other objects and advantages of the present invention will becomeapparent from the following description, taken in connection with theaccompanying figures and examples, wherein, by way of illustration andexample, an embodiment of the present invention is disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

IN THE DRAWINGS

FIG. 1 shows prior art aminoglycosides;

FIG. 2 shows prior art target sites of modifying enzymes;

FIG. 3 shows some exemplary structures of compounds according to thepresent invention;

FIG. 4 shows structures of some exemplary designed acceptors accordingto the present invention;

FIG. 5 shows a general outline of a synthetic scheme according to thepresent invention;

FIG. 6 shows preparation of an acceptor according to the presentinvention;

FIG. 7 shows some exemplary donor structures according to the presentinvention;

FIG. 8 shows an exemplary synthetic scheme for producing a library ofstructures;

FIG. 9 shows two sets of exemplary neomycin derivatives according to thepresent invention;

FIG. 10 shows an exemplary synthetic scheme according to the presentinvention;

FIG. 11 shows an exemplary synthetic scheme for glycosyl donors 5 e, 5 cand 8 b according to the present invention;

FIG. 12 shows the synthesis of some compounds and intermediatesaccording to the present invention;

FIG. 13 shows synthesis of another exemplary group of intermediatesaccording to the present invention;

FIG. 14 shows an exemplary synthesis of set6 structures according to anembodiment of the present invention;

FIG. 15 shows an additional exemplary synthesis of set6 structuresaccording to an embodiment of the present invention; and

FIG. 16 shows the structures of neomycin B (Compound I) and CompoundsII-XIII according to the present invention.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention is of a novel group of aminoglycosides which sharesome structural features of currently available aminoglycosides withregard to the backbone, while also having significant structuraldifferences. The similarity enables these aminoglycosides to beeffective antibiotics, whilezzz the significant differences enable theseaminoglycosides to reduce or even block antibiotic resistance. Theaminoglycosides of the present invention are suitable for inhibition ofanthrax lethal factor, hence are suitable for use as a cure for anthrax.

The anthrax lethal factor (LF), a Zn-dependent endopeptidase, has amajor role in the development and virulence of anthrax. The compounds ofthe present invention have been found to act as powerful inhibitors ofthe proteolytic activity of LF at seemingly physiological conditions andsimultaneously function as antibiotics against Bacillus anthracis.

To find novel inhibitors of LF, a library of approximately 3000compounds were tested, over 60 of which were synthetic and commercialaminoglycosides (23). While a number of the tested compoundsdemonstrated some level of inhibitory activity, neomycin B was found tobe the most potent inhibitor of LF with apparent K_(i) value in the lownM concentration range.

The compounds of the present invention were obtained through rationaldesign of antibiotics, based upon known aminoglycosides. However, unlikethe previously described rational design strategies, which have manysignificant drawbacks with regard to the potency and/or side effects ofthe designed compounds, the compounds of the present invention weredesigned according to a new and better strategy. Without wishing to belimited by a single hypothesis, it would appear that sinceaminoglycoside antibiotics, such as neomycin B, exert theirantibacterial activity by selectively recognizing and binding to thedecoding A site on the 16S subunit of the bacterial rRNA, causingdeleterious misreading of the genetic code (24). At physiological pH,aminoglycosides are highly charged and their RNA binding relies onelectrostatic interactions (25-26). Examination of the recentlydetermined X-ray crystal structure of LF shows that the active site ofthe protease also consists of a broad, 40 Å groove with a highlynegative electrostatic potential (27).

Docking experiments were performed, which showed that neomycin B couldreside within the vicinity of the catalytic zinc, and multiple potentialcontacts could occur between the negatively charged residues of LF andneomycin B (23).

Based on these data, it was hypothesized that since the interaction ofneomycin B with both the rRNA and LF is mainly determined byelectrostatic interactions, it is likely that by maintaining theantibiotic backbone intact but adding one or more additionalrecognition/binding elements, superior binding to both rRNA and LF, andprobably better antibacterial performance is expected to result.Enhanced RNA binding by using dimerized aminoglycosides (26),bifunctional aminoglycosides (27), and amino-aminoglycosides (28), alongwith the inhibition of various nucleic acids metabolizing enzymes byaminoglycosides (29), support this hypothesis.

The compounds of FIG. 16 keep the whole antibiotic constitution intactas a recognition element to both the rRNA and LF. The extended sugarring(s) of each structure was designed in a manner that incorporatesdifferent combinations of hydroxyl and amino groups as potentialfunctionalities directed for the recognition of the phosphodiester bondof rRNA (30) and in parallel the Asp/Glu and Asn/Gln clusters in theactive site of LF (23).

The principles and operation of the compositions, processes and methodsaccording to the present invention may be better understood withreference to the Examples and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

The compounds of the present invention are based upon neomycin B(Compound I, FIG. 3) as a base structure, with three sets of designedbifunctional mimetics (set1-set3, FIG. 3).

Preferred compounds according to the present invention can becollectively represented by general Formula I:

wherein:

-   R₁ is a monosaccharide residue or an oligosaccharide residue;-   X and Y are independently oxygen or sulfur;-   R₂ and R₃ are each independently selected from the group consisting    of hydrogen, hydroxy, thiol, amine, alkyl, cycloalkyl, aryl, alkoxy,    aryloxy, thioalkoxy and thioaryloxy, as these terms are defined    hereinabove; and    wherein the carbon at the fifth position of ring B has an R    configuration or an S configuration;    and pharmaceutically acceptable salts thereof.

Preferably X is oxygen. Also preferably, Y is oxygen. Optionally andpreferably, R₁ is a monosaccharide residue. More preferably, themonosaccharide residue is a five-membered (furanose) or a six-membered(pyranose) monosaccharide residue. Also more preferably, themonosaccharide residue comprises at least one amine group and/or atleast one aminoalkyl group. Optionally and more preferably, the at leastone amine group and/or the at least one aminoalkyl group is at one ormore of positions 2, 3, 4 or 5. As used herein, the term “aminoalkyl”refers to an alkyl group, as defined hereinabove, which is substitutedby an amine group, as defined hereinabove. Optionally, at least oneaminoalkyl group is an aminomethyl group (CH₂—NH₂).

Optionally and preferably, if the monosaccharide residue is a pyranosemonosaccharide residue, the aminomethyl group is at position 5.

Also optionally and preferably, if the monosaccharide residue is apyranose monosaccharide residue, the amine group is at one or more ofpositions 2, 3 or 4.

Also optionally and preferably, if the monosaccharide residue is afuranose monosaccharide residue, the aminoalkyl group is at position 4.

Optionally, the monosaccharide residue is a L-monosaccharide or aD-monosaccharide.

According to preferred embodiments of the present invention, R₁ is anoligosaccharide residue. Preferably, the oligosaccharide residuecomprises at least two monosaccharide residues, wherein each isindependently a five-membered (furanose) or a six-membered (pyranose)monosaccharide residue. More preferably, at least one of the at leasttwo monosaccharide residues comprises at least one amine group and/or atleast one aminoalkyl group. Most preferably, the at least one aminegroup is at position 2 of a pyranose monosaccharide residue. Also mostpreferably, the at least one aminoalkyl group is at position 5 of apyranose monosaccharide residue.

Optionally and preferably, the oligosaccharide comprises a furanosemonosaccharide linked to a pyranose monosaccharide. .Optionally, each ofthe at least two monosaccharide residues is independently aD-monosaccharide or a L-monosaccharide.

Optionally, R₁ comprises an oligosaccharide residue consisting of atleast four monosaccharide residues, whereby each of the monosaccharideresidues is a a five-membered (furanose) or a six-membered (pyranose)monosaccharide residue.

Such oligosaccharide residues can be, for example, an aminoglycosideresidue such as a Neomycin B residue, a Paromomycin residue, aRibostamycin residue, a Gentamycin residue, a Amikacin residue, aNeamine residue, a Nebramine residue and a Tobramine residue.

Such oligosaccharide residues preferably further compirse a freechemical group that is coupled to X in Formula I above. Preferably, bothX and the chemical group in the oligomers residue are S, such that theolidosacchride residue is coupled via a disulfide bond.

According to other preferred embodiments of the present invention, X issulfur and R₁ is a monosaccharide residue. Preferably, themonosaccharide is a furanose monosaccharide residue.

According to still other preferred embodiments of the present invention,there are provided novel compounds each having the general formula II:

wherein:

-   Y is oxygen or sulfur;-   R₂ and R₃ are each independently hydrogen, hydroxy, thiol, amine,    alkyl, cycloalkyl, aryl, alkoxy, aryloxy, thioalkoxy and    thioaryloxy; and    wherein the carbon at the fifth position of ring B has an R    configuration or an S configuration;

and pharmaceutically acceptable salts thereof.

Preferably, Y is oxygen, and R₂ and R₃ are both hydroxy.

According to still other preferred embodiments of the present invention,there are provided novel compounds each having the general formula III:

wherein:

-   X is disulfide, as defined hereinabove;-   Y is oxygen or sulfur;-   R₁ is an oligosaccharide residue having at least four monosacchride    residues, as described hereinabove;-   R₂ and R₃ are each independently hydrogen, hydroxy, thiol, amine,    alkyl, cycloalkyl, aryl, alkoxy, aryloxy, thioalkoxy and    thioaryloxy, as these terms are defined hereinabove; and    wherein the carbon at the fifth position of ring B has an R    configuration or an S configuration;

and pharmaceutically acceptable salts thereof.

It should be noted that wherever reference is made to a general formulaor a specific compound according to the present invention,pharmaceutically acceptable salts are also optionally included.

All of the different structures of the preferred compounds according tothe present invention are shown in FIG. 16.

In selecting the modification site in neomycin B and the degree ofmodification, recent structural information has been included in thedesign process, again without wishing to be limited by a singlehypothesis, as follows. Superposition of neomycin B bound to theaminoglycoside kinase APH(3′) ternary complex with ADP (10), andparomomycin I (contains C6′—OH instead of C6′—NH₂ in neomycin B, FIG. 1)bound to A-site bacterial ribosome (25) reveals that all the functionalgroups of aminoglycosides that are utilized for binding are identical inboth antibiotics, with the exception of two groups, which are notemployed for binding in the antibiotic-resistance enzyme.

One of these different groups is the C5″—OH of neomycin B which isphased towards the second substrate, ATP, and may have a crucial rolefor the formation of the reactive ternary complex prior to occurrence ofthe phosphorylation step. Therefore, without wishing to be limited by asingle hypothesis, incorporation of gross changes, such as the additionof extra rigid sugar ring in this region, is expected to have a dramaticeffect on the formation of a precise ternary complex required forenzymatic catalysis. For a number of reasons, including the abovehypothesis and also ease of synthesis, position C5″ in neomycin B wasselected as the base for the new generation of pseudo-pentasaccharidesof set1 (FIG. 3), with the expectation that they will function betterthan neomycin B against both the resistant and non-resistant organisms.

These structures maintain the antibiotic backbone intact as arecognition element to the rRNA, while the extended sugar ring (R₁) ineach structure is designed in a manner that incorporates either plainpyranose sugar, a single amino group at various positions,cis-1,2-diamine, flexible 1,3-diamine, cis-1,3-hydroxyamine, orribofuranose ring as potential functionalities directed for therecognition of the phosphodiester bond of RNA (29-31) (For the detailedstructures at ring R₁ see structures of the designed donors in FIG. 7).

The designed structures of set2 (FIG. 3) are similar to that of set1except for the sulfur atom at C5″. These structures were speciallydesigned, again without wishing to be limited by a single hypothesis, toavoid in vivo enzymatic hydrolysis of the added sugars (ring E) byvarious exoglycosidases (especially in those structures in which ring Econtain either plain sugar or 2-aminohexose). The structures of set3 aresimilar to that of set1 except for the ring A. In the structures ofset3, ring A is 3′,4′-dideoxy sugar, again without wishing to be limitedby a single hypothesis, to combat against the action of various APH(3′)enzymes.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLE I General Synthesis of the Compounds of the Present Inventionand Syntheses of Specific Exemplary Intermediates

The strategy for the construction of all three sets of compounds in FIG.3 featured the use of a common acceptor for each set (acceptors 1-3 inFIG. 4), to which the monosaccharide donors were connected, followed bya two-step deprotection to yield the target C5″-branched derivatives.The protecting groups used in this study served admirably in terms ofease of attachment and removal and survivability under the reactionconditions, whereas the thioglycoside-NIS (31) andtrichloroacetimidate-BF₃ (32) glycosidation methods proved to be bothrapid and efficient.

This Example describes the overall synthetic procedure with optionalvariations; the following Examples include specific non-limitingexamples of the synthetic process as it was performed for the presentinvention.

The neomycin acceptor 1 is readily accessible from the commercialneomycin B (49). The C5″—SH acceptor 2 can easily be prepared from theselectively protected hexaazido derivative of neomycin B (acceptor 1) intwo steps as outlined in FIG. 5. Briefly, acceptor 2 was prepared asfollows: Triphenylphosphine (1.153 grams, 4.4 mmol) was dissolved in dryTHF (10 mL) under argon and was stirred at 0° C. for 15 minutes. Themixture was then added dropwise with diisopropylazodicarboxylate (0.627mL, 4.4 mmol). The mixture was stirred for 45 minutes at 0° C. and awhite precipitate of the betaine was observed. In an additional flaskacceptor 1 (1.5 gram, 1.467 mmol) and thioacetic acid (0.23 mL, 4.4mmol) were dissolved in THF (4 mL) under argon, and added dropwise in tothe flask containing the betaine. Propagation of the reaction wasmonitored by TLC (EtOAc 50%, Hexane 50%), which indicated completionafter 4.5 hours. The mixture was diluted with EtOAc and washed withbrine. The combined organic layer was dried over MgSO₄, evaporated andpurified by column chromatography (silica, EtOAc/Hexane) to yield thecorresponding thioacetate 2 a as white solid 1.36 gram (86%).

¹H NMR (500 MHz, CDCl₃) data of this thioacetate are summarized in Table12 hereinbelow.

¹³C NMR: δ=20.4, 20.6, 20.7, 20.9, 31.0 (C-2), 31.2 (C-5″), 50.5 (C-6″),50.9 (C-6′), 56.6, 57.9, 59.1, 60.6, 65.5, 68.7, 69.1, 69.2, 69.9, 73.1,74.9, 75.2, 75.7, 77.8, 80.2, 81.4, 96.2 (C-1′), 99.9 (C-1′″), 105.6(C-1″), 168.5, 169.5, 169.7, 169.9, 170.0, 195.1

ESIMS: m/z=1119.3 (M+K⁺, C₃₇H₄₈N₁₈O₁₉S requires 1119.5).

The pure thioacetate 2 a from the above (250 mg, 0.231 mmol) wasdissolved in dry DMF under argon, and added with hydraziniumacetate(42.6 mg, 0.463 mmol). Propagation of the reaction was monitored by TLC(EtOAc 50%, Hexane 50%), which indicated completion after 3 hours. Themixture was diluted with EtOAc and washed with brine. The combinedorganic layer was dried over MgSO₄, evaporated and purified by columnchromatography (silica, EtOAc/Hexane) to yield the thiol acceptor 2 aswhite solid 156 mg (65%).

¹H NMR (500 MHz, CDCl₃) data of acceptor 2 are summarized in Table 13hereinbelow.

¹³C NMR: δ=20.4, 20.5, 20.6, 20.7, 26.5 (C-5″), 31.3 (C-2), 50.6(C-6′″), 50.8(C-6′), 56.3, 57.9, 59.0, 60.5, 65.5, 68.6, 69.2, 69.7,73.4, 75.0, 75.3, 75.9, 77.1, 81.3, 81.6, 96.2 (C-1′), 99.0 (C-1′″),106.3 (C-1″), 168.4, 169.4, 169.6, 169.6, 169.9, 169.9

MALDI-TOFMS: m/z=1077.0 (M+K⁺, C₃₅H₄₆N₁₈O₁₈S requires 1077.6).

The dideoxy acceptor 3 can be prepared from compound 4 (FIG. 6) byselective protection of C3′ and C4′ hydroxyls by cyclohexylidene,followed by acetylation, selective removal of cyclohexylidene, andtwo-step simultaneous deoxygenation of C3′ and C4′ hydroxyls accordingthe reported procedure (32). As an alternative to acetate protectionwhich may not be stable under Bu₃SnH treatment, benzyl protection isused.

These newly designed acceptors can be represented by the general formulaIV:

wherein:

each of Z₁ and Z₂ is independently selected from the group consisting ofhydrogen, alkyl, cycloalkyl, aryl, a hydroxy protecting group, an aminoprotecting group and a thiol protecting group; each of T₁-T₄ isindependently a hydroxy protecting group; each of Q₁-Q₆ is independentlyan amino protecting group;

X is oxygen or sulfur; Y is oxygen or sulfur; and

wherein the carbon at the fifth position of ring B has an Rconfiguration or an S configuration.

As is exemplified hereinabove, the hydroxy protecting group can be, forexample, an O-acetyl group, whereas the amino protecting group can be,for example, an azido group.

As used herein, the phrase “an O-acetyl group” refers to a —O—C(═O)CH₃group, in which the hydroxy group is protected by an acetyl group.

The phrase “an azido group” refers to a —N₃ group, in which the aminogroup is protected by an azo group.

However, other hydroxy and amino protecting groups commonly used inchemical syntheses in general and in saccharide syntheses in particularare also usable in this context of the present invention.

Acceptors 1-3, according to the present invention, are compounds havingthe general formula IV above, wherein, for acceptor 1, X is oxygen, eachof Z₁, Z₂ and OT₁-OT₄ is an O-acetyl group and each of NQ₁-NQ₆ is anazido group; for acceptor 2, X is sulfur, each of Z₁, Z₂ and OT₁-OT₄ isan O-acetyl group and each of NQ₁-NQ₆ is an azido group; and, foracceptor 3, X is oxygen, each of Z₁, Z₂ is hydrogen, each of OT₁-OT₄ isan O-acetyl group and each of NQ₁-NQ₆ is an azido group.

The donors in FIG. 7 were designed as thioglycosides since thethioglycoside-NIS glycosidation method proved to be both rapid andefficient. The N-phth and ester protections at C-2 of the monosaccharidedonors were designed to allow, through neighboring group participation,selective β-glycoside bond formation between rings E and C (33-34).

The donors, according to preferred embodiments of the present invention,are therefore compounds having the general formula V, VI or VII:

wherein each of G, I, J, K, U and V is independently selected from thegroup consisting of a hydroxy protecting group (e.g., O-acetyl group,O-chloroacetyl group, and O-benzoyl group) and an amino protecting group(e.g., an azido group and a N-phtalimido group); SL is a thiolatedleaving group (e.g., thioethyl and para-thiotoluene); and each of thecarbons at positions 1, 3 and 4 in Formula I and at position I inFormula II has an R configuration or an S configuration.

The overall synthesis of each of the pseudo-pentasaccharides of thepresent invention having the general formula I above is thereforeeffected, according to preferred embodiments of the present invention,by:

(i) providing an acceptor having the general formula IV describedhereinabove;

(ii) providing a donor having the general formula V, VI or VII;

(iii) coupling the acceptor and the donor, to thereby provide aprotected pseudo-pentasaccharide; and

(iv) removing the protecting groups, to thereby provide the desiredcompound.

Preferably, the assembly of the designed protectedpseudo-pentasaccharides (set1-set3) is performed by NIS-promotedcoupling of each of the acceptors 1-3 with the thioglycoside donors (5a-f, 6 a-f, 7 a-c, and 8 a-d, FIG. 7). The resulting protected compoundsare then subjected to a two-step deprotection process: removal of allthe ester and phtalimido groups by treatment with methylamine (33%solution in EtOH) and reduction of all the azido groups by Staudingerreaction, to furnish a library of 57 pseudo-pentasaccharide derivativesof neomycin B, as generally illustrated in FIG. 8. Note that for thepreparation of set2 structures, the thioglycoside donors are convertedto the corresponding trichloroacetimidates and the coupling steps areperformed under acidic conditions (BF₃-Et₂O.CH₂Cl₂).

The overall synthesis of each of the compounds of the present inventionhaving the general formula II described above is effected, according topreferred embodiments of the present invention, by:

(i) providing an acceptor having the general formula IV, as describedhereinabove, wherein X is sulfur; and

(ii) removing the protecting groups.

The overall synthesis of each of the compounds of the present inventionhaving the general formula III described above is effected, according topreferred embodiments of the present invention, by:

(a) providing a compound having the general formula IV, as describedhereinabove, wherein X is preferably sulfur;

(b) providing an oligosaccharide comprised of at least fourmonosaccharide residues and having at least one free thiol groupattached to at least one of the monosaccharide residues, wherein anyhydroxy group or amino group attached to the monosaccharide resiudes isprotected by a hydroxy protecting group or an amino protecting group,respectively;

(c) coupling the compound having general formula IV with theoligosaccharide, to form a disulfide bond therebetween; and

(d) removing each of the hydroxy protecting groups and amino protectinggroups, to provide the compound of general formula III.

The reacting oligosaccharide can thus be a compound having the generalformula IV, such that this synthesis results is a Neomycin B “dimer” inwhich the two units are linked by a disulfide bond. Alternatively, thereacting oligosaccharide can be, for example, Neamine, Tobramine,Tobramycin, or Gentamycin, which can preferably be modified as describedherein with respect to acceptors 1-3 so as to have a free thiol group.The hydroxy or amino protecting groups, present in cases where theoligosaccharide bears hydroxy or amino groups, are as described herein.

Coupling is preferably effected by Lewis acid (BF₃-Et₂O) promotedcoupling, which after two-steps deprotection as above provided thedesired thioglycoside.

EXAMPLE 2 Selection of Structures for Compounds of the Present Invention

The previous Example related to a general scheme which may optionally beused for any compound according to the present invention, as well asoptionally for generating a library of compounds according to thepresent invention. This Example describes the selection of somenon-limiting, illustrative structures for compounds according to thepresent invention.

One important aspect of the present invention is the use of functionalaminoglycosides to solve the problem of cytotoxicity. Without wishing tobe limited by a single hypothesis, these structures were selected toameliorate this problem. One of the major drawbacks of aminoglycosidesis their relatively high toxicity. Neomycin B is the most toxic ofaminoglycosides, yet it is primarily used for topical infections. It ishighly nephrotoxic and ototoxic and is by far the most potent in thearea of neuromuscular blockage. Aminoglycosides are nephrotoxic becausea small but sizable proportion of the administered dose (about 5%) isretained in the epithelial cells (35). Aminoglycosides accumulated bythese cells are mainly localized with endosomal and lysosomal vacuolesbut are also localized with the Golgi complex, causing an array ofmorphological and functional alterations of increasing severity. It isalso believed that aminoglycosides cause the formation of free radicals,which lead to cell death (36).

Very recently (37), however, it has been shown that aminoglycosidesstabilize DNA and RNA triplexes. A clear correlation between thetoxicity (LD₅₀ values, the lethal dose, or dose sufficient to kill halfthe test population) of these antibiotics and their ability to stabilizeDNA triple helix was demonstrated and suggested that aminoglycosides maybe able to aid H-DNA formation in vivo, which might be one of thereasons for their toxicity. Interestingly, these results also showedthat neomycin B, which is most toxic among all aminoglycosides, is alsothe most active of all aminoglycosides in stabilizing triple helices,and that neomycin B does not influence the double helical structures ofDNA structures. Paromomycin (FIG. 1), which differs from neomycin B inthat it has one less amino group,. is much less toxic than neomycin B(LD₅₀ of neomycin=24 mg/kg, paromomycin=160 mg/kg). Thus, thisdifference of one charge makes a great difference in the toxicity of thetwo compounds. Further deletion of charged amino groups in ribostamycinmakes it least toxic (LD₅₀ of ribostamycin=260 mg/kg). On the otherhand, lividomycin, which differs from paromomycin by an additionalmannose, is much less toxic, with a LD₅₀ value of 280 mg/kg. From thesedata it seems that two factors that significantly reduce the toxicity ofaminoglycoside are: reduction of the number of amino groups and/oraddition of an extra saccharide.

Without wishing to be limited by a single hypothesis, the above featuresof aminoglycosides and their different levels of cytotoxicity wereconsidered when selecting suitable structures for the compounds of thepresent invention. Since neomycin B is at the “head of the peak” withlowest LD₅₀ value, this structure was selected for designing two sets ofderivatives, set4 and set5 (FIG. 9). The rationale in designing set4structures is to generate new analogs of neomycin B, “thio-neomycins,”with superior acid stability, which can lead to a reduction in therequired dose for administration and subsequently a lowering of theassociated toxicity. The choice of the thioglycosidic linkage betweenthe rings B and C in set4 structures is based on the fact that theglycosidic bond of a furanose is more acid sensitive than that ofpyranose. Indeed, this is the reason that neomycin B and all the membersof neomycin family suffer a high acid sensitivity. To solve thisproblem, Chang and co-workers (12f) have recently reported on the newclass of “pyranmycins” in which the furanose ring of ribostamycin hasbeen replaced by various pyranose structures. Some of the resultedpseoudo-trisaccharides have indeed showed increased acid stability andsubstantial antibacterial activity. The suggested production of the“thio-neomycins” is an improved, elegant solution to this problem.

The rationale behind the design of set5 structures is largely based onthe recent structural information obtained by Fong and Berghuis (10).This work has shown that while the conformation of aminoglycosides andthe functional groups utilized for the binding are effectively identicalwhen comparing the neomycin B-bound structure of APH(3′)-IIIa and theparomomycin I-bound structure of the 30S ribosome, there are significantdifferences when examining the van der Waals interactions. The moststriking difference found is that the face of the aminoglycoside thatforms most of the van der Waals interactions with APH(3′)-IIIa isopposite to that which interacts with the 16S ribosomal RNA.

Without wishing to be limited by a single hypothesis, this observationwas used to design novel variant(s) of neomycin B which can interactwith the ribosome A-site but which are unable to be detoxified byAPH(3′)-IIIa and related enzymes. Examples of these variants accordingto the present invention include the set5 structures, “epi-neomycins”.In these structures, the configuration of neomycin is inverted at C5,which is the branch point between two parts of the molecule, rings A-Band C-D. Such an inversion of configuration in neomycin is expected toresult in broad effects. Without wishing to be limited by a singlehypothesis, the change of orientation at C5 (from equatorial in neomycinto axial in epi-neomycin) should allow more rotational freedom betweenrings A and B, and between A-B and C-D. Consequently, but again withoutwishing to be limited by a single hypothesis, the face of the resultedepi-neomycins is expected to be preferentially recognized by rRNA, whileit will be highly hindered for the recognition byaminoglycoside-modifying enzymes. In addition, the resultedconformational changes in set5 structures, relative to neomycin, shouldaffect the stabilization of DNA triple helix and subsequently decreasethe toxicity of this set of compounds. Optionally, the two concepts ofthese structures may be combined to obtain variants of“thio-epi-neomycins.”

EXAMPLE 3 Specific Synthesis of Selected Compounds of the PresentInvention

Example 1 included a general synthetic scheme which may optionally beused for any compound according to the present invention, as well asoptionally for generating a library of compounds according to thepresent invention. This Example provides an illustrative, non-limitingsynthetic process that was performed for selected compounds according tothe present invention.

As shown in FIGS. 10-12, a compound was prepared according to asynthetic scheme which started with neomycin B being converted to ageneral acceptor, as described with regard to Example 1. FIGS. 10 and 11show the syntheses of the monosaccharide donors. Neomycin B (Compound I)is shown in FIG. 12 after being converted to an acceptor 1 to which themonosaccharide donors of FIG. 7 can be coupled.

The protecting groups used in this study served admirably in terms ofthe ease of attachment and removal and survivability under the reactionconditions, whereas the thioglycoside-NIS glycosidation method(Veeneman, G. H.; van Leeuwen, S. H.; van Boom, J. H. Tetrahedron Lett.1990, 31, 1331-1334) proved to be both rapid and efficient. The N-phthand ester protections at C-2 of the monosaccharide donors were designedto allow, through neighboring group participation, selective, glycosidebond formation between rings E and C.

For FIG. 10, the following reagents and conditions were used: for stagea, (i) TBDPSCl, pyridine, DMAP, 60° C.; (ii) 2,2-dimethoxypropane,acetone, CSA; (iii) BzCl, pyridine, DMAP; (iv) AcOH/H₂O 9:1, THF, 60°C., 55% for four steps. For stage b, (i) Tf₂O, pyridine; (ii) NaN₃, DMF,HMPA, 63% for two steps; (iii) HF/pyridine; (iv) ClAcCl, pyridine, 91%for two steps. For stage c, (i) Anisaldehyde-dimethylacetal, CSA, THF;(ii) BzCl, pyridine; (iii) AcOH/H₂O 9:1, THF, 60° C., 58% for threesteps. For stage d, (i) Tf₂O, pyridine; (ii) NaN₃, DMF, HMPA, 86% fortwo steps.

As a general note for all procedures described herein (unless otherwisenoted), reactions were monitored by TLC on Silica Gel 60 F254 (0.25 mm,Merck), and spots were visualized by charring with a yellow solutioncontaining (NH₄)Mo₇O₂₄.4H₂O (120 grams) and (NH₄)₂Ce(NO₃)₆ (5 grams) in10% H₂SO₄ (800 mL). Flash column chromatography was performed on SilicaGel 60 (70-230 mesh). All reactions were carried out under an argonatmosphere with anhydrous solvents, unless otherwise noted. Allchemicals unless otherwise stated, were obtained from commercialsources.

The diazido monosaccharides 9 and 5 f, having D-allo and D-glucoconfigurations, respectively, were constructed from the commonD-galactose derivatives (FIG. 10) by selectively inverting theconfigurations at C3 and C4 (in 9) and at C4 (in 5 f). Briefly, the diol14 was prepared from the known thioglycoside 12 (Pozsgay, V.; Jennings,H. J. Tetrahedron Lett. 1987, 28, 1375-1376) in four steps (selectivesilylation of the primary hydroxyl, acetonide formation at C3—OH andC4—OH, benzoylation, and removal of the acetonide) without isolation ofintermediate products in an overall yield of 55%.

More specifically, ethyl2-O-benzoyl-6-O-tert-butyldiphenylsilyl-1-thio-β-D-galactopyranoside(compound 14) was prepared from ethyl2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyranoside (Pozsgay, V.;Jennings, H. J. Tetrahedron Lett. 1987, 28, 1375-1376), by using thefollowing five steps procedure.

To a suspension of ethyl2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyranoside (6.25 grams, 16mmol) in dry MeOH (70 mL) and dry dichloromethane (70 mL) was addedcatalytic amount of NaOMe (0.5M solution in MeOH) at 0° C.. Propagationof the reaction was monitored by TLC (MeOH 10%, dichloromethane 90%).After 2 hours the reaction mixture was neutralized by Dowex H+ andevaporated to dryness. The resultant crude preparation of 12 (3.2 grams,14.3 mmol) was used for the next step without further purification.

The crude of 12 from the previous step (3.2 grams, 14.3 mmol) in drypyridine (35 mL) was added with a catalytic amount of DMAP and stirredunder argon at 60° C. for 10 minutes. The mixture was added withtert-butyldiphenylsilylchloride (6.7 mL, 25.7 mmol) and the reactionprogress was monitored by TLC (EtOAc 65%, Hexane 35%). After 30 minutesthe mixture was diluted with EtOAc and washed with brine, 1.5% H₂SO₄,NaHCO₃ (saturated), and finally with brine. The organic layer was driedover MgSO₄ and evaporated to give a pale yellow syrup (8.2 grams) thatwas used for the next step without further purification.

The crude from the previous step (8.2 grams) in acetone (50 mL) and2,2-dimethoxypropane (25 mL) was stirred at ambient temperature for 5minutes and then added with a catalytic amount of camphorsulfonic acid.Propagation of the reaction was monitored by TLC (EtOAc 30%, Hexane70%). After 3 hours the reaction mixture was neutralized by NH₄OH (2.5%)and evaporated to dryness. The crude was diluted with EtOAc and washedwith brine. The combined organic layer was dried over MgSO₄ andevaporated to give a pale yellow syrup (8.7 grams).

The crude from the previous step (8.7 grams) was added with a catalyticamount of DMAP in dry pyridine (50 mL) and stirred at ambienttemperature for 5 minutes. The reaction mixture was added withbenzoylchloride (4.14 mL, 35.7 mmol) and the propagation of the reactionwas monitored by TLC (Et₂O 20 percentages, Hexane 80%). After 4 hoursthe mixture was diluted with EtOAc and the organic phase was washed asfollows: brine, HCl (2%), NaHCO₃ (sat.) and brine. The combined organiclayer was then dried over MgSO₄ and evaporated to give a pale yellowsyrup (9.3 grams).

The crude from the previous step (9.3 grams) was dissolved in THF (10mL), AcOH (50 mL) and water (5 mL). The reaction mixture was stirred at60° C. for 5 hours. Propagation of the reaction was monitored by TLC(EtOAc 30%, Hexane 70%). The reaction mixture was diluted with EtOAc andthe organic phase was neutralized with NaHCO₃ (sat.), and washed withbrine. The combined organic layer was dried over MgSO₄, evaporated andpurified by flash chromatography (silica, EtOAc/Hexane) to yieldcompound 14 as pale-yellow syrup (4.34 grams, 48% yield for the fivesteps).

¹H NMR (500 MHz, CDCl₃): δ=1.05 (s, 9H, t-Bu), 1.25 (t, 3H, J=7.5 Hz,SCH₂CH₃), 2.68 (m, 2H, SCH₂CH₃), 3.59 (m, 1H, H-5), 3.76 (dd, 1H, J₁=3,J₂=9.5 Hz, H-3), 3.97 (m, 2H, H′-6, H-6), 4.14 (d, 1H, J=2.18 Hz, H-4),5.01 (d, 1H, J=10 Hz, H-1), 5.14 (t, 1H, J=9, Hz, H-2), 7.38-8.12 (m,15H, aromatic).

¹³C NMR (125 MHz, CDCl₃): δ=26.8(C(CH₃)₃), 63.7(C-6), 69.7(C-4),72.2(C-2), 74.2(C-3), 78.0(C-5), 80.1(C-1), 127.6, 127.7, 128.6, 129.7,129.8, 130.1, 133.7, 135.6, 135.8.

ESIMS: m/z=605.1 (M+K^(+ C) ₃₁H₃₈O₆SSi requires 605.3).

Simultaneous triflation of both hydroxyls in 14 was followed bynucleophilic displacement with azide (without isolation of theintermediate ditriflate) to afford the corresponding diazide (63%isolated yield for two steps). Desilylation was then followed with achloracetylation step to produce the allo-donor 9. Ethyl2-O-Benzoyl-3,4-dideoxy-3,4-diazido-6-O-chloroacetyl-1-thio-β-D-allopyranoside(compound 9) was prepared as follows. Compound 14 (1.68 gram, 2.97 mmol)was dissolved in dichloromethane (8 mL) and pyridine (0.73 mL, 7.4mmol), and was stirred at 0° C. for 10 minutes. To this mixture wasadded Tf₂O (1.08 mL, 6.4 mmol) and the propagation of the reaction wasmonitored by TLC (EtOAc 20%, Hexane 80%), which indicated completionafter 15 minutes. In an additional flask a mixture of NaN₃ (3.795 grams,58.3 mmol), dry DMF, (40 mL) and HMPA (5 mL) was vigorously stirredunder argon, and added at once in to the reaction mixture. The reactionwas heated to 50° C., and propagation was monitored by TLC (EtOAc 20%,Hexane 80%). After 3 hours the mixture was diluted with EtOAc and washedwith brine, HCl (2%), NaHCO₃ (sat.), brine. The combined organic layerwas dried over MgSO₄, evaporated and purified by flash chromatography(silica, Diethylether/Hexane) to yield the corresponding 3,4-diazidoproduct as a pale yellow syrup (1.15 gram, 63%).

¹H NMR (500 MHz, CDCl₃): δ=1.07 (s, 9H, t-Bu), 1.24 (t, 3H, J=9.5,SCH₂CH₃), 2.68 (m, 2H, SCH₂CH₃), 3.73 (broad d, 1H, J=9.5, Hz, H-5),3.88 (dd, 1H, J₁=3.0, J₂=12.0 Hz, H-6), 3.97 (d, 1H, J=11.5 Hz, H-6′),4.51 (dd, 1H, J₁=3.0, J₂=6.0 Hz, H-3), 4.06 (dd, 1H, J₁=3.0, J₂=9.5 Hz,H-4), 4.95 (d, 1H, J=9.5 Hz, H-1), 5.19 (dd, 1H, J₁=3.0, J₂=10, H-2),7.38-8.11 (m, 15H, aromatic).

¹³C NMR (125 MHz, CDCl₃): δ=26.8 (C(CH₃)₃), 57.7 (C-4), 63.0 (C-6), 63.1(C-3), 70.2 (C-2), 75.7 (C-5), 79.6 (C-1), 127.6, 127.7, 128.6, 129.7,129.8, 130.1, 133.7, 135.6, 135.8.

ESIMS: m/z=655.2 (M+K^(+ C) ₃₁H₃₆N₆O₄SSi requires 655.3).

The di-azido product (1.15 gram, 1.87 mmol) was dissolved in pyridine (4mL) and stirred in a polyethylene vessel at 0° C. for 10 minutes. Themixture was added with HF/Pyr (4 mL) and its propagation was monitoredby TLC (EtOAc 20%, Hexane 80%). After 5 minutes the mixture was dilutedwith EtOAc and neutralized with NaHCO₃ (sat.). The combined organiclayer was dried over MgSO₄ and evaporated to dryness. The residue wasdissolved in pyridine (10 mL) and added with a catalytic amount of DMAP,followed by the addition of chloroacetylchloride (0.286 mL, 3.73 mmol).Propagation was monitored by TLC (EtOAc 20%, Hexane 80%). After 25minutes the mixture was diluted with EtOAc, washed with brine, HCl (2%),NaHCO₃ (sat.), and brine. The combined organic layer was dried overMgSO₄, evaporated and purified by flash chromatography (silica,EtOAc/Hexane) to yield the titled compound 9 as a pale yellow syrup (777mg, 91%).

¹H NMR (500 MHz, CDCl₃): δ=1.23 (t, 3H, J=10.0, SCH₂CH₃), 2.68 (m, 2H,SCH₂CH₃), 3.73 (dd, 1H, J₁=3.0, J₂=10.0 Hz, H-4), 3.94 (ddd 1H, J₁=2.5,J₂=4.5, 1H, J₃=10.0 Hz, H-5), 4.1 (s, 2H, COCH₂Cl) 4.29 (dd, 1H, J₁=5,J₂=12.5 Hz, H-6), 4.51 (m, 2H, H-3,H-6), 4.97 (d, 1H, J=10 Hz, H-1),5.16 (dd, 1H, J₁=3.5, J₂=10.0 Hz, H-2), 7.44-8.08 (5H, aromatic).

¹³C NMR (125 MHz, CDCl₃): δ=15 (SCH₂CH₃), 24.2 (SCH₂CH₃), 40.6(COCH₂Cl), 58 (C-4), 62.5 (C-3), 64.7 (C-6), 70.0 (C-2), 72.6 (C-5),80.4 (C-1), 127.6, 127.7, 128.6, 129.7, 129.8, 130.1, 133.7, 135.6,135.8.

ESIMS: m/z=493.5 (M+K⁺ C₁₇ClH₁₉N₆ _(O) ₅S requires 493.6).

Alternatively, selective protection of C6 and C4 hydroxyls in thegalactoside 13 by p-methoxybenzylidene, followed by benzoylation andhydrolysis of the benzylidene, gave the diol 15 in an overall 58% yieldfor three steps.

p-Methylphenyl 2,3-Di-O-benzoyl-1-thio-β-D-galactopyranoside (compound15) was prepared as follows: To a solution ofp-methylphenyl-1-thio-β-D-galactopyranoside (Zhang, Z.; Ollmann, I. R.;Ye, X-S.; Wischnat, R.; Baasov, T.; Wong, C-H. J. Am. Chem. Soc., 1999,121, 734.) (4.7 g, 16 mmol) in DMF (30 mL) and anisaldehydedimethylacetal (3.6 mL, 21 mmol) was added a catalytic amount ofcamphorsulfonic acid and stirred at ambient temperature. The reactionwas monitored by TLC (EtOAc 70%, Hexane 30%). After 2 hours the mixturewas diluted with EtOAc, and washed with brine, NaHCO₃ (sat.), and onceagain with brine. The combined organic layer was dried over MgSO₄,evaporated and purified by flash chromatography (silica, EtOAc/Hexane)to yield the corresponding 4,6-O-benzylidene (4.9 grams, 76%).

¹H NMR (125 MHz, CDCl₃): δ=2.47 (s, 3H, Me-STol), 3.62 (broad s, 1H, Hz,H-5), 3.75 (dd 1H, J₁ =J ₂=9.5 Hz, H-2), 3.79 (dd, 1H, J₁=3, J₂=9.5 Hz,H-3), 3.92 (s, 3H, Me-OMP), 4.10 (d, 1H, J=11.5 Hz, H-6), 4.28 (d, 1H,J=3 Hz, H-4) 4.45 (d, 1H, J=11.5 Hz, H-6′), 4.10 (d, 1H, J=9.5 Hz, H-1),5.55 (s, 1H, Benzylic Proton), 6.96-7.70 (m, 8H, aromatic).

¹³C NMR (125 MHz, CDCl₃): δ=21.2 (Me-STol), 55.2 (Me-OMP), 68.6, 69.2,69.9, 73.7, 75.3, 87, 101.1(C-1), 113.4, 127.8, 129.6, 131.9, 132.1,134.2, 138.3.

Positive CIMS: m/z=405.1 (M+H⁺ C₂₁H₂₄O₆S requires 404.2).

The product from the previous step (4.9 grams, 12.2 mmol) was dissolvedin dry pyridine under argon and added with a catalytic amount of DMAP.After being stirred at ambient temperature for 5 minutes, the reactionmixture was added with benzoylchloride (3.7 mL, 31.4 mmol). Propagationof the reaction was monitored by TLC (EtOAc 30%, Hexane 70%), whichindicated completion after 4 hours. The reaction mixture was dilutedwith EtOAc and the organic phase was washed as follows: brine, HCl (2%),NaHCO₃ (sat.), brine. The combined organic layer was dried over MgSO₄,and evaporated to dryness to yield 6.3 grams of crude that was used forthe next step without further purification.

The crude from the previous step (6.3 grams) was added with THF (10 mL),AcOH (50 mL), and water (5 mL). The reaction mixture was stirred at 50°C. for 3 hours. Propagation of the reaction was monitored by TLC (EtOAc30%, Hexane 70%). The reaction mixture was diluted with EtOAc and theorganic phase was neutralized by NaHCO₃ (sat.), and washed with brine.The combined organic layer was dried over MgSO₄, evaporated and purifiedby flash chromatography (silica, EtOAc/Hexane) to yield compound 15(4.62 grams, 58% for the three steps).

¹H NMR (500 MHz, CDCl₃/CD₃OD 10/1): δ=2.21 (s, 3H, Me-STol), 3.50-3.90(m, 3H, H-5, H-6, H-6′), 4.27 (d, 1H, J=3.5 Hz, H-4), 4.80(d, 1H, J=9.9Hz, H-1), 5.17 (dd, 1H, J₁=4.5, J₂=9.9 Hz, H-3), 5.64 (t, 1H, J=4.2 Hz,H-2) 6.98-7.90(m, 14H, aromatic).

¹³C NMR (125 MHz, CDCl₃): δ=20.9 (Me-STol), 61.4, 67.3, 68.2, 75.6,78.5, 87.2(C-1), 128.2, 128.8, 129.3, 129.6, 132.6, 133.1, 133.2, 138.1,165.5, 166.0.

Negative CIMS: m/z=494.3 (M+H⁺ C₂₆H₂₀O₇S requires 495.3).

This diol (compound 15) was then subjected to a similar triflation andazidation steps as for compound 14 to afford the 4,6-diazido donor 5 fin an isolated yield of 86% for two steps.

p-Methylphenyl-4,6-Dideoxy-4,6-diazido-2,3-O-benzoyl-1-thio-β-D-glucopyranoside(compound 5 f) was prepared as follows. Compound 15 (4.62 grams, 9.35mmol) in dry pyridine (20 mL) was stirred under argon at 0° C. for 10minutes and added with Tf₂O (1.97 mL, 11.7 mmol). The mixture wasallowed to warm to room temperature. Propagation of the reaction wasmonitored by TLC (EtOAc 20%, Hexane 80%), which indicated completionafter 15 minutes. In an additional flask, a mixture of NaN₃ (12.17grams, 187 mmol) in dry DMF, (40 mL) and HMPA (5 mL) was vigorouslystirred under argon, and added at once into the reaction mixture.Propagation was monitored by TLC (EtOAc 20%, Hexane 80%), whichindicated completion after 3 hours. The mixture was diluted with EtOAcand washed with brine, HCl (2%), NaHCO₃ (sat.), and brine. The combinedorganic layer was dried over MgSO₄, evaporated and purified by flashchromatography (silica, EtOAc/Hexane) to yield compound 5 f (3.66 grams,72 percentages yield).

¹H NMR (500 MHz, CDCl₃): δ=2.33 (s, 3H, Me-STol), 3.51 (dd, 1H, J₁=4.5,J₂=13.5 Hz, H-6), 3.56 (ddd 1H, J₁=1.5, J₂=4.5, 1H, J₃=15.0 Hz, H-5),3.70 (dd, 1H, J₁=1.5, J₂=13.0 Hz, H-6′), 3.83 (t, 1H, J=9.5 Hz, H-4),4.82 (d, 1H, J=10.0 Hz, H-1), 5.26 (t, 1H, J=9.5 Hz, H-2), 5.61 (d, 1H,J=9.5 Hz, H-3), 7.10-7.95 (m, 14H, aromatic).

¹³C NMR (125 MHz, CDCl₃): δ=21.2 (Me-STol), 51.5 (C-6), 60.5 (C-4), 70.1(C-2), 74.9 (C-3), 77.4 (C-5), 86.1 (C-1), 126.6, 128.4, 128.5, 129.0,129.7, 129.8, 133.4, 133.5, 134.6, 134.7, 139.1, 165.0, 165.6.

ESIMS: m/z=583.1 (M+K⁺ C₂₇H₂₄O₅N₆S requires 583.3).

Ethyl3,4-Di-O-benzoyl-6-O-chloroacetyl-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranose(compound 10) was prepared by the same synthetic path which was used forthe preparation of phenyl6-O-acetyl-3,5-di-O-benzoyl-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranose(Solomon, D.; Fridman, M.; Zhang, J.; Baasov, T. Organic Letters 2001,3, 4311-4314).

Ethyl 2,3,5-Tri-O-acetyl-1-thio-D-ribofuranose (compound 11) wasprepared as follows. The commercial1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (3.5 grams, 11 mmol) indichloromethane (30 mL) was added with ethylthiotrimethylsilane (4.44mL, 27.5 mmol) and TMSOTf (2 mL, 11 mmol). The mixture was stirred atambient temperature and the reaction progress was monitored by TLC(EtOAc/Hexane 1:1), which indicated completion after 3.5 hours. Themixture was diluted by EtOAc (200 mL), neutralized by NaHCO₃ (sat.), andwashed with brine. The combined organic layer was dried over MgSO₄,evaporated and purified by flash chromatography (silica, EtOAc/Hexane)to yield compound 11 3.4 grams, (96% yield) as a mixture of anomers(α/β; 1:3).

¹H NMR (500 MHz, CDCl₃) for 9-α-anomer: δ=4.07 (dd, 1H, J₁=4.0, J₂=11.5Hz, H-5), 4.17 (dd, 1H, J₁=4.0, J₂=9.5 Hz, H-4), 4.28 (dd, 1H, J₁=3.0,J₂=12.0 Hz, H-5), 5.09 (d, 1H, J₁=3.0 Hz, H-1), 5.16 (dd, 1H, J₁=J₂=5.0Hz, H-2), 5.26(dd, 1H, J₁=J₂=5.5 Hz, H-3).

¹³C NMR (125 MHz, CDCl₃): δ=14.7, 20.37, 20.4, 20.7, 24.5, 63.5 (C-5),71.6, 74.4, 79.5, 85.5 (C-1), 169.4, 169.5, 170.3.

¹H NMR (500 MHz, CDCl₃) for 9-α-anomer: δ=4.12 (dd, 1H, J₁3.5, J₂=12.0Hz, H-5), 4.25(dd, 1H, J₁=3.5, J₂=12.0 Hz, H-5′), 4.27 (dd, 1H, J₁=4.0,J₂=8.0 Hz, H-4), 5.08 (t, 1H, J=6.0 Hz, H-3), 5.30 (d, 1H, J=6.5 Hz,H-2), 5.53 (d, 1H, J=4.5 Hz, H-1). ¹³C NMR (125 MHz, CDCl₃) , 14.9,20.3, 20.4, 20.6, 25.3, 62.8 (C-5), 70.4, 71.3, 74.4, 86.7 (C-1), 169.3,169.7, 170.4.

¹³C NMR (125 MHz, CDCl₃): δ=21.2 (Me-STol), 51.5 (C-6), 60.5 (C-4), 70.1(C-2), 74.9 (C-3), 77.4 (C-5), 86.1 (C-1), 126.6, 128.4, 128.5, 129.0,129.7, 129.8, 133.4, 133.5, 134.6, 134.7, 139.1, 165.0, 165.6.

Negative CIMS: m/z=319.1 (M−H⁺ C₁₃H₂₀O₇S requires 320.1).

p-Methylphenyl-2-deoxy-2-phthalimido-6-deoxy-6-azido-3,4-di-O-benzoyl-1-thio-β-D-glucopyranoside(5 e) was prepared fromp-Methylphenyl-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside (Wong,Chi-Huey; Zhang, Zhiyuan; Ollmann, Ian; Baasov, Timor; Ye, Xin-Shan, J.Am. Chem. Soc, 1999, 121, 734-753.), by the following four steps:

p-Methylphenyl-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside (2grams, 4.8 mmol) in dry pyridine (35 mL), was added with a catalyticamount of DMAP and stirred under argon at 60° C. for 10 minutes. Themixture was added with tert-butyldiphenylsilylchloride (2.51 mL, 9.63mmol) and the reaction progress was monitored by TLC (EtOAc 65%, Hexane35%). After 2 hours the mixture was allowed to cool back to roomtemperature, and added with benzoyl chloride (1.67 mL, 14.45 mmol) andthe propagation of the reaction was monitored by TLC (EtOAc 40%, Hexane60%). After 4 hours the mixture was diluted with EtOAc and the organicphase was washed as follows: brine, HCl (2%), NaHCO₃ (sat.) and brine.The combined organic layer was then dried over MgSO₄ and evaporated toafford a pale yellow syrup.

The crude from the previous step was dissolved in pyridine (15 mL) andstirred under argon at 0° C. for 10 minutes in a polyethylene vessel.The mixture was added with HF/Pyr (15 mL) and its propagation wasmonitored by TLC (EtOAc 20%, Hexane 80%). After 5 minutes the mixturewas diluted with EtOAc and neutralized with NaHCO₃ (sat.). The combinedorganic layer was dried over MgSO₄, evaporated to dryness and purifiedby flash chromatography (silica, EtOAc/Hexane) to yield the titledcompound as 2.52 grams (84% for the 3 steps).

¹H NMR (200 MHz): δ=1.63 (broad s, 1H, 6—OH), 2.31 (s, 3H, SPhCH₃), 3.69(dd, 1H, J₁=4.7, J₂=12.7 Hz, H-6), 3.82-3.93 (m, 2H, H-5, H-6′), 4.54(t, 1H, J=10.4 Hz, H-2), 5.46 (t, 1H, J=9.8 Hz, H-4), 5.81 (d, 1H,J=10.6 Hz, H-1), 6.28 (t, 1H, J=10.2, Hz, H-3), 6.91-7.92 (m, 18H,aromatic).

Positive CIMS: m/z=623.3 (M⁺ C₃₅H₂₉NO₈S requires 623.1).

The pure alcohol from the above (1.5 gram, 2.4 mmol) was dissolved inpyridine (30 mL), and was stirred at 50° C. for 10 minutes followed bythe addition of freshly crystallized p-toluenesulfonyl chloride (1.15gram, 6.01 mmol). Propagation of the reaction was monitored by TLC(EtOAc 20%, Hexane 80%), which indicated completion after 15 minutes.The mixture was diluted with EtOAc and the organic phase was washed asfollows: brine, HCl (2%), NaHCO₃ (sat.) and again with brine. Thecombined organic layer was then dried over MgSO₄ and evaporated toafford a pale yellow syrup. The tosylation product was then put underargon and added NaN₃ (1.562 gram, 24 mmol), dry DMF (40 mL) and HMPA (5mL). The reaction was heated to 50° C., and propagation was monitored byTLC (EtOAc 20%, Hexane 80%). After 3 hours the mixture was diluted withEtOAc and washed with brine, HCl (2%), NaHCO₃ (sat.), brine. Thecombined organic layer was dried over MgSO₄, evaporated and purified byflash chromatography (silica, EtOAc/Hexane) to yield 5 e as white solid(2.23 grams, 93% yield).

¹H NMR (500 MHz, CDCl₃): δ=2.36 (s, 3H, SPhCH₃), 3.44 (dd, 1H, J₁=2.5,J₂=13.5 Hz, H-6), 3.50 (dd, 1H, J₁=6.0, J₂=13.5 Hz, H-6′), 4.06 (ddd,1H, J₁=3.0, J₂=6.5, J₃=13.0 Hz H-5), 4.55 (t, 1H, J=10.5 Hz, H-2), 5.48(t, 1H, J=9.5 Hz, H-4), 5.83 (d, 1H, J=10.5 Hz, H-1), 6.23 (t, 1H,J=10.0, Hz, H-3), 7.13-7.90 (m, 18H, aromatic).

¹³C NMR (125 MHz, CDCl₃): δ=21.2 (SPhCH₃), 51.4, 53.7, 70.3, 71.9, 77.4,83.4, 123.7, 128.3, 128.4, 128.5, 129.8, 131.2, 131.6, 134.2, 134.3,134.5, 139.1, 165.2, 165.6, 166.9, 168.0.

ESIMS: m/z=687.4 (M+K⁺ C₃₅H₂₈N₄O₇S requires 687.2).

p-Methylphenyl-5-deoxy-5-azido-2,3-di-O-benzoyl-1-thio-D-ribofuranose (8b) was prepared from the commercially available1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (Sigma) by the following 5steps:

1,2,3,5-Tetra-O-acetyl-β-D-ribofuranose (3 grams, 9.43 mmol) in drydichloromethane (35 mL) was added with 4-methylbenzenethiol (1.4 gram,11.78 mmol), treated with TMSOTf (0.35 mL, 1.925 mmol) and stirred atambient temperature under argon. Propagation of the reaction wasmonitored by TLC (EtOAc/Hexane 1:1), which indicated completion after 11hours. The mixture was diluted by EtOAc (200 mL), neutralized by NaHCO₃(sat.), and washed with brine. The combined organic layer was dried overMgSO₄, evaporated and used for the next step without furtherpurification.

A suspension of the crude from the previous step in dry MeOH (40 mL) anddry dichloromethane (40 mL) was added a catalytic amount of NaOMe (0.5Msolution in MeOH) at 0° C.. Propagation of the reaction was monitored byTLC (MeOH 10%, dichloromethane 90%). After 2 hours the reaction mixturewas neutralized by Dowex H⁺ and evaporated to dryness. The resultedcrude was used for the next step without further purification.

The crude from the previous step was dissolved under argon in drydichloromethane (60 mL) and added with dry triethylamine (3 mL). Themixture was then added with freshly crystallized p-toluenesulfonylchloride (2.16 grams, 11.31 mmol) and stirred 4° C.. Propagation of thereaction was monitored by TLC (MeOH 10%, dichloromethane 90%) andindicated completion after 12 hours. The mixture was then evaporated todryness and added NaN₃ (1.562 gram, 24 mmol), dry DMF (30 mL) and HMPA(3 mL). The reaction was heated to 50° C., and propagation was monitoredby TLC (EtOAc 40%, Hexane 60%). After 3 hours the mixture was dilutedwith EtOAc and washed with brine, HCl (2%), NaHCO₃ (sat.), brine. Thecombined organic layer was dried over MgSO₄, evaporated to dryness toafford the crude product as a pale orange colored oil.

The crude from the previous step was dissolved in dry pyridine (40 mL)under argon. The mixture was added with benzoyl chloride (3.27 mL, 27.4mmol) and propagation was monitored by TLC (EtOAc 30%, Hexane 70%).After 4 hours, the mixture was diluted with EtOAc and washed with brine,HCl (2%), NaHCO₃ (sat.), brine. The combined organic layer was driedover MgSO₄, evaporated and purified by column chromatography (silica,EtOAc/Hexane) to yield 8 b 2.63 grams, (57% yield for the five steps) asa mixture of anomers (α/β; 1:4).

¹H NMR (500 MHz, CDCl₃) for the β-anomer: δ=2.36 (s, 3H, Me-STol), 3.58(d, 2H, J=4.5 Hz, H-5, H-5′), 4.40 (dd, 1H, J₁=4.5, J₂=9.5 Hz, H-4),5.47 (t, 1H, J=5.5 Hz, H-3), 5.34 (s, 1H, H-1), 5.58(t, 1H, J=5.0 Hz,H-2), 7.17-8.11(17.5H. aromatic protons of both anomers).

¹³C NMR (125 MHz, CDCl₃): δ=21.1 (Me-Stol), 52.7(C-5), 72.5(C-3),74.6(C-2), 81.6(C-4), 88.6(C-1), 128.4, 128.8, 128.9, 129.8, 129.9,130.6, 165.0, 165.3.

For the α-anomer ¹H NMR (500 MHz, CDCl₃): δ=2.33 (s, 3H, Me-STol), 3.65(dd, 1H, J₁=4.0, J₂=13.0 Hz, H-5), 3.75(dd, 1H, J₁=3.0, J₂=13.5 Hz,H-5′), 4.67 (dd, 1H, J₁=4.0, J₂=7.0 Hz, H-4), 5.59 (dd, 1H, J₁=4.5,J₂=5.5 Hz, H-3), 5.74 (t, 1H, J=6.0 Hz, H-2), 6.03 (d, 1H, J=6.0 Hz,H-1) 7.17-8.11(17.5H. aromatic protons of both anomers).

¹³C NMR (125 MHz, CDCl₃): δ=21.1 (Me-Stol), 51.8(C-5), 71.8(C-3),72.2(C-2), 80.2(C-4), 90.9(C-1), 127.7, 128.5, 128.9, 129.0, 129.7,138.8, ESIMS m/z 528.3 (M+K³⁰C₂₆H₂₃N₃O₅S requires 528.5).

p-Methylphenyl-6-O-Acetyl-4-deoxy-4-azido-3,4-di-O-benzoyl-1-thio-β-D-glucopyranoside(5 c) was prepared fromp-Methylphenyl-2,3-di-O-benzoyl-1-thio-β-D-galactopyranoside 15 by thefollowing four steps:

p-Methylphenyl-2,3-di-O-benzoyl-1-thio-β-D-galactopyranoside (900 mg,1.82 mmol) in dry pyridine (12 mL), was added with a catalytic amount ofDMAP and stirred under argon at 50° C. for 10 minutes. The mixture wasadded with tert-butyldiphenylsilylchloride (1.07 mL, 4.09 mmol) and thereaction progress was monitored by TLC (EtOAc 50%, Hexane 50%). After 30minutes, the mixture was diluted with EtOAc and washed with brine, HCl(2%), NaHCO₃ (sat.), brine. The combined organic layer was dried overMgSO₄, evaporated and purified by column chromatography (silica,EtOAc/Hexane) to yield the product as white solid 1.1 gram (82% yield).

¹H NMR (500 MHz, CDCl₃): δ=1.06 (s, 9H, tert-butylSiPh₂), 2.30 (s, 3H,SPhCH₃), 3.76 (t, 1H, J=5.5 Hz, H-5), 3.93 (dd, 1H, J₁=4.5, J₂=15.5 Hz,H-6), 4.02 (dd, 1H, J₁=4.5, J₂=16.0 Hz, H-6′), 4.45 (d, 1H, J=3.0 Hz,H-4), 4.85 (d, 1H, J=10.0 Hz, H-1), 5.28 (dd, 1H, J₁=3.0, J₂=10.0 Hz,H-3), 5.78 (t, 1H, J=9.5 Hz, H-2) , 6.98-7.98 (24H, aromatic).

¹³C NMR (125 MHz, CDCl₃): δ=21.2 (SPhCH₃), 26.8 (tert-butylSiPh₂), 64.0,68.0, 68.5, 75.8, 77.9, 86.9 (C-1), 127.8, 127.9, 128.3, 128.4, 128.6,129.2, 129.6, 129.8, 129.9, 132.7, 133.1, 133.3, 135.6, 135.7, 138.2,165.2, 165.9.

ESIMS: m/z=771.2 (M+K⁺ C₄₃H₄₄O₇SiS requires 771.8).

The product of the previous step (1 gram, 1.36 mmol) was dissolved inpyridine (8 mL), and stirred at 0° C. for 15 minutes followed by thedropwise addition of trifluoromethanesulfonic anhydride (0.26 mL, 1.564mmol). Propagation of the reaction was monitored by TLC (EtOAc 20%,Hexane 80%), which indicated completion after 15 minutes. The mixturewas evaporated under vacuum to afford a pale yellow syrup, and was thenput under argon and added NaN₃ (1.848 gram, 28 mmol), dry DMF (30 mL)and HMPA (5 mL) and stirred at room temperature. Propagation wasmonitored by TLC (EtOAc 15%, Hexane 85%). After 10 hours and the mixturewas diluted with EtOAc and washed with brine, HCl (2%), NaHCO₃ (sat.),brine. The combined organic layer was dried over MgSO₄ evaporated andused for the next step without further purification.

The crude from the previous step was dissolved in pyridine (8 mL) andstirred under argon at 0° C. for 10 minutes in a polyethylene vessel.The mixture was added with HF/Pyr (4 mL) and its propagation wasmonitored by TLC (EtOAc 25%, Hexane 75%). After 5 minutes the mixturewas diluted with EtOAc and neutralized with NaHCO₃ (sat.). The combinedorganic layer was dried over MgSO₄, evaporated to dryness and used forthe next step without further purification.

The crude from the previous step was dissolved in pyridine (8 mL). Themixture was then added with a catalytic amount of 4-DMAP and aceticanhydride (0.258 mL, 2.8 mmol) with HF/Pyr (4 mL) and its propagationwas monitored by TLC (EtOAc 20%, Hexane 80%). After 2 hours the mixturewas diluted with EtOAc and neutralized with NaHCO₃ (sat.). The combinedorganic layer was dried over MgSO₄, evaporated to dryness and used forthe next step without further purification. The mixture was diluted withEtOAc and washed with brine, HCl (2%), NaHCO₃ (sat.), brine. Thecombined organic layer was dried over MgSO₄, evaporated and purified bycolumn chromatography (silica, EtOAc/Hexane) to yield the 5 c as whitesolid 615 mg (73% yield for the four steps).

¹H NMR (500 MHz, CDCl₃): δ=2.14 (s, 3H, OCOMe), 2.32 (s, 3H, SPhCH₃),3.63 (ddd, 1H, J₁=2.0, J₁=4.5, J₁=10.0 Hz, H-5), 3.79 (t, 1H, J=10.0 Hz,H-4), 4.30 (dd, 1H, J₁=5.0 J₂=12.5 Hz, H-6), 4.52 (dd, 1H, J₁=2.0,J₂=12.0 Hz, H-6′), 4.45 (d, 1H, J=3.0 Hz, H-4), 4.80 (d, 1H, J=9.5 Hz,H-1), 5.29 (t, 1H, J=9.5 Hz, H-2), 5.60.(t, 1H, J=9.5 Hz, H-3),7.07-7.94 (14H, aromatic).

¹³C NMR (125 MHz, CDCl₃): δ=20.8.7 (SPhCH₃), 21.2 (OCOMe), 60.5, 63.0,70.3, 79.4, 76.2, 76.9, 86.3 (C-1), 127.6, 128.4, 128.7, 129.1, 129.7,129.8, 129.9, 133.4, 133.5, 134.0, 138.8, 165.1, 165.6, 170.5.

ESIMS: m/z=600.1 (M+K⁺ C₂₉H₂₇N₃O₇S requires 600.7).

Turning now to FIG. 12, the neomycin acceptor 1 was readily prepared infour chemical steps from the commercial neomycin B (Compound I) in anoverall yield of 57% (briefly, these steps included perazidation of thecommercial neomycin B (obtained from Sigma Israel) with TfN₃ accordingto the procedure of Wong (Kumar, V.; Jones, G. S., Jr.; Blacksberg, I.;Remers, W. A. J. Med. Chem. 1980, 23, 42-49; Yoshikawa, M.; Ikeda, Y.;Takenaka, K. Chem. Lett. 1984, 13, 2097-2100), selective silylation ofthe primary hydroxyl at C5″, acetylation of all the remaining hydroxyls,and desilylation as depicted in FIG. 12).

The neomycin acceptor 1 was prepared as follows: Hexaazido-neomycin wasprepared from the commercial neomycin B (tri-sulfate salt, 5 grams, 5.5mmol) following the published procedure (Alper, P. B.; Hendrix, M.;Sears, P.; Wong, C.-H. J. Am. Chem. Soc. 1998, 120, 1965-1978) and wasused for the next step without purification. The crude hexaazido-productwas dissolved in pyridine (40 mL), added with DMAP (cat.) and stirred at70° C. for 15 minutes. The reaction was then added withtert-butyldimethylsilylchloride (1.66 gram, 11 mmol), and TLC (EtOAc100%) indicated completion after 30 minutes. The mixture was allowed tostir for additional 10 minutes and then added with pyridine (20 mL),DMAP (cat), and Ac₂O (7.8 mL, 82.5 mmol). Propagation of the reactionwas monitored by TLC (EtOAc 30%, Hexane 70%), which indicated completionafter 3 hours. The mixture was diluted with EtOAc and washed with brine,HCl (2%), NaHCO₃ (sat.), and brine. The combined organic layer was driedover MgSO₄, evaporated and purified by flash chromatography (silica,EtOAc/Hexane) to yield the corresponding silyl ether as a white powder(3.5 grams, 62% yield for 3 steps).

¹H NMR (500 MHz, CDCl₃) data for this compound is summarized in Table 1hereinbelow.

¹³C NMR (150.92 MHz): δ=18.2, 20.5, 20.6, 20.7, 20.9, 25.8, 29.6, 31.4,50.7, 51.1, 56.7, 58.1, 59.2, 60.8, 63, 65, 68.7, 69, 70, 73.1, 75.2,76, 76.7, 76.9, 77, 77.2, 81.6, 83.3, 96.0 (anomeric carbon), 99.6(anomeric carbon), 106.3 (anomeric carbon), 168.5, 169.5, 169.7, 167.9,170.2, 170.3.

ESIMS: m/z=1175.6 (M+K⁺, C₄₁H₆₀O₁₉N₁₈Si requires 1175.4).

The silyl ether from the above (1.06 gram, 0.93 mmol) was dissolved inpyridine (8 mL) and stirred in a polyethylene vessel at 0° C. for 10minutes. The mixture was added with HF/Pyr (4 mL). Propagation of thereaction was monitored by TLC (EtOAc 20%, Hexane 80%), which indicatedcompletion after 5 minutes. The mixture was diluted with EtOAc,neutralized with NaHCO₃ (sat.). The combined organic layer was driedover MgSO₄, evaporated and purified by flash chromatography (silica,EtOAc/Hexane) to yield acceptor 1 as a white powder (884 mg, 93% yield).

¹H NMR (500 MHz, CDCl₃) data of acceptor 1 is summarized in Table 2hereinbelow.

¹³C NMR: δ=15.9, 16, 22.3, 22.4, 22.5, 22.6, 33.3, 52.6, 52.7, 58.3,59.7, 60.8, 62.1, 67.4, 70.5, 70.9, 71, 71.1, 75.2, 77.8, 83.2, 83.6,99.0 (anomeric carbon), 101.2 (anomeric carbon), 108.0 (anomericcarbon), 170.4, 171.2, 171.4, 171.6, 171.7, 171.9.

ESIMS: m/z=1061.2 (M+K⁺, C₃₅H₄₆O₁₉N₁₈ requires 1061.4).

NIS-promoted coupling of acceptor 1 with various thioglycosidesfurnished the designed protected pseudo-pentasaccharides 16 a-h in58-89% yields. Purity and exclusive stereochemistry of new glycosidicbonds in 16 a-d were confirmed by ¹H NMR spectroscopy (16 a: H-1, δ 4.86ppm, J_(1,2)=8.0 Hz. 16 b: H-1, δ 4.81 ppm, J_(1,2)=7.5 Hz. 16 c: H-1, δ5.62 ppm, J_(1,2)=8.5 Hz. 16 d: H-1, δ 5.95 ppm, J_(1,2)=4.5 Hz; seebelow for a more detailed description and tables).

Compound 16 a was prepared as follows: To powdered, flame dried 4 Åmolecular sieves (0.4 gram) was added CH₂Cl₂ (4 mL), followed by theaddition of acceptor 1 (100 mg, 0.098 mmol) and donor 9 (65 mg, 0.143mmol). After being stirred for 10 minutes at room temperature, themixture was treated with NIS (64.3 mg, 0.286 mmol). After an additional5 minutes at room temperature, TfOH (cat.) was added. Propagation of thereaction was monitored by TLC (EtOAc 50%, Hexane 50%), which indicatedcompletion after 10 minutes. The reaction was diluted with EtOAc, andfiltered through celite. After thorough washing with EtOAc, the washeswere combined and extracted with 10% Na₂S₂O₃, saturated (aq.) NaHCO₃,brine, dried over MgSO₄ and concentrated. The crude was purified byflash chromatography to yield 16 a (112 mg, 81% yield).

¹H NMR (500 MHz, CDCl₃) data of 16 a are summarized in Table 3hereinbelow.

¹³C NMR: δ=20.4, 20.6, 20.7, 29.6, 31, 40.7, 50, 50.9, 56.3, 58, 58.8,60.6, 6.60, 62.5, 65, 68.1, 68.4, 69, 70.3, 71, 72.4, 74.8, 75, 75.5,76.4, 80.2, 83.5, 96.4 (anomeric carbon), 98.8 (anomeric carbon), 99.0(anomeric carbon), 108.2 (anomeric carbon), 128.9, 129.3, 129.7, 133.7,167.7, 167.1, 168.3, 169.5, 169.6, 169.8, 170.1.

ESIMS: m/z=1453.2 (M⁺K⁺, C₅₀ClH₅₉ N₂₄O₂₄ requires 1453).

Compound 16 b. The titled compound was prepared as was described for thepreparation of compound 16 a. The conditions used were: donor 5 f (430mg, 0.79 mmol), acceptor 1 (646 mg, 0.63 mmol), NIS (430 mg, 1.9 mmol),TfOH (cat.) CH₂Cl₂ (15 mL), and 4 Å molecular sieves (1.5 gram). Thereaction was performed at 0° C. to yield 16 b (780 mg, 86%) as a mixtureof anomers (α/β 1:2) as determined by NMR.

ESIMS: m/z=1481.7 (M⁺K⁺, C₅₅H₆₂N₂₄O₂₄ requires 1481.6).

The above mixture could not be separated until after the next step.Thus, compound 16 b (200 mg, 0.138 mmol, as a mixture of anomers) wasdissolved in 33% solution of MeNH₂ in EtOH (40 mL) and stirred at roomtemperature for 48 hours. Propagation of the reaction was monitored byTLC (MeOH 20%, CHCl₃ 80%). The reagent and the solvent were removed byevaporation and the residue was purified by flash chromatography(silica, MeOH/CHCl₃) to yield the corresponding octaazido-octaol as aβ-anomer 16 bβ(72.7 mg, 53%), and the octaazido-heptaol-C_(2V)-O-benzoylas an α-anomer 16 bα(49.2 mg, 36%).

¹H NMR (500 MHz, CDCl₃/CD₃OD; 10:1) data of the β-anomer and of theα-anomer are summarized in Table 4 and Table 5 hereinbelow,respectively.

¹³C NMR (β-anomer): δ=29.5, 38.1, 51.1, 51.2, 51.5, 59.6, 59.8, 60.6,62.3, 62.5, 68.7, 68.9, 69.4, 71.1, 71.1, 73.6, 73.6, 73.7, 74.1, 75.3,75.4, 75.9, 76.1, 80.6, 84.3, 96.2 (anomeric carbon), 98.3 (anomericcarbon), 102.8 (anomeric carbon), 108.5 (anomeric carbon).

ESIMS: m/z=1021.3 (M+K⁺, C₂₉H₄₂N₂₄O₁₆ requires 1021.8).

¹³C NMR (β-anomer): δ=29.5, 39.1, 51.0, 51.4, 51.7, 59.4, 59.7, 60.9,62.9, 62.9, 68.5, 69.7, 70.8, 71.0, 71.2, 74.1, 75.0, 75.6, 75.9, 76.2,80.6, 84.2, 96.5 (anomeric carbon), 97.7 (anomeric carbon), 98.4(anomeric carbon), 107.3 (anomeric carbon), 120.1, 125.9, 128.3, 129.4,135.9.

ESIMS: m/z=1125.3 (M⁺K⁺, C₃₆H₄₇N₂₄O₁₇ requires 1125.8).

Compound 16 c. The titled compound was prepared as described for thepreparation of compound 16 a. The conditions used were: donor 10 (467.2mg, 0.73 mmol), acceptor 1 (250 mg, 0.24 mmol), NIS (330 mg, 1.47 mmol),TfOH (cat.). CH₂Cl₂ (5 mL), 4 Å molecular sieves (500 mg), to yield 226mg of 16 c (58%).

¹H NMR (500 MHz, CDCl₃) data of 16 c are summarized Table 6 hereinbelow.

¹³C NMR: δ=20.4, 20.7, 20.8, 20.9, 29.6, 40.8, 50.5, 54.7, 56.6, 58.2,59.0, 60.7, 65.5, 69.0, 69.8, 70.2, 70.8, 72.0, 73.0, 75.1, 75.3, 76.4,76.9, 81.0, 81.2, 96.4 (anomeric carbon), 98.6 (anomeric carbon), 99.5(anomeric carbon), 107.2 (anomeric carbon), 128.3, 128.4, 128.5, 128.6,129.7, 129.9, 133.3, 133.5, 165.2, 165.7, 167.1, 168.4, 169.5, 169.7,169.8, 169.9, 170.1.

ESIMS: m/z=1636.3(M+K⁺, C₆₅H₆₈N₁₉O₂₈Cl requires 1637.1).

Compound 16 d: The titled compound was prepared as was described for thepreparation of compound 16 a with the following modifications: Donor 11(157 mg, 0.49 mmol), acceptor 1 (200 mg, 0.196 mmol), NIS (110 mg, 0.49mmol), TfOH (cat.), 4 Å molecular sieves (400 mg). In an attempt toincrease the beta selectivity, acetonitrile (4 mL) was used as a solventand the reaction temperature was −35° C. Under these conditions, 16 dwas isolated as a mixture of anomers α/β; 1:11 (238 mg, (95%). Thismixture was further separated to afford 178 mg (71%) of the pureβ-anomer.

¹H NMR (500 MHz, CDCl₃) data of 16 d are summarized in Table 7hereinbelow.

¹³C NMR: d=20.4, 20.6, 20.7, 20.8, 23.8, 29.5, 29.7, 50.6, 50.7, 56.7,57.9, 59.2, 60.43, 60.86, 62.1, 63.44, 64.45, 65.62, 68.6, 68.9, 69.2,70.0, 70.7, 70.9, 71.2, 72, 73.2, 75.2, 75.3, 76.1, 77.3, 77.8, 78.5,79.1, 81.3, 81.7, 97.9 (anomeric carbon), 101.4 (anomeric carbon), 106.1(anomeric carbon), 108.5 (anomeric carbon), 170.4, 171.5, 171.6, 171.8,171.9, 172.5.

ESIMS: m/z=1319.3(M+K⁺, C₄₆H₆₀N₁₈O₂₆ requires 1319.5).

These protected compounds (16 a-d) were subjected to a two-stepdeprotection, removal of all the ester and phthalimido groups bytreatment with methylamine (33% solution in EtOH) and reduction of allthe azido groups by Staudinger reaction, to furnish the final products,compounds II-V, with high purity and isolated yields, as described ingreater detail below after the preparation of compounds 16 e-h.

Compound 16 i: To a powdered, flame dried 4 Å molecular sieves (0.7gram) was added CH₂Cl₂ (7 mL) containing donor 8 e (298 mg, 0.491 mmol)(prepared according to the published: Ivan Chiu-Machado, Julio CCastro-Palomino, Madrazo-Alonso, Carlos Lopetegui-Palacios and VicenteVeers-Bencomo J. Carbohydr. Chem. 1995, 14, 551-561.) and acceptor 2(127.6 mg, 0.123 mmol). After being stirred for 20 minutes at roomtemperature, the mixture was cooled to −10° C. and treated with BF₃OEt₂(40 μL). Propagation of the reaction was monitored by TLC (EtOAc 45%,Hexane 55%), which indicated completion after 45 minutes. The reactionwas quenched with triethylamine, diluted with EtOAc, and filteredthrough celite. After thorough washing of the celite with EtOAc, thewashes were combined and extracted with saturated (aq.) NaHCO₃, brine,dried over MgSO₄ and concentrated. The crude was purified by flashchromatography (silica, EtOAc/Hexane) to yield 16 i compound (164 mg,90% yield).

¹H NMR (500 MHz, CDCl₃) data of 16 i are summarized in Table 14hereinbelow.

¹³C NMR: δ=20.5, 20.6, 20.7, 20.9, 31.3 (C-2), 33.6 (C-5″), 50.5(C-6′″), 51.0 (C-6′), 60.75, 64.3 (C-5″″), 65.6, 68.8, 69.2, 69.5, 70.0,72.5, 73.7, 74.4, 75.3, 76.2, 76.3, 78.1, 79.4, 80.9, 81.1, 86.9(C-1″″), 96.4 (C-1′), 98.9 (C-1′″), 106.3 (C-1″), 128.4, 128.5, 128.6,128.8, 129.1, 129.6, 129.7, 129.8, 128.9, 133.1, 133.4, 133.6, 165.1,165.2, 166.1, 168.5, 169.5, 169.6, 169.8, 170.1, 170.1.

MALDI-TOFMS: m/z=1522.0 (M+K⁺, C₆₁H₆₆N₁₈O₂₅S requires 1521.9).

Compound 19 a was prepared according to the procedure published bySwayze et al. (Baogen Wu, Jun Yang, Yun He and Eric E. Swayze, OrganicLetters, 2002, 4(20), 3455-3458). Acceptor 1 (250 mg, 0.2445 mmol) wasdissolved in dry CH₂Cl₂ (9.8 mL) under argon, and added with4-methylbenzenethiol (33.1 mg, 0.269 mmol) and stirred at 0° C. for 15minutes. The mixture was then treated with BF₃OEt₂ (0.093 mL, 0.733mmol), stirred at 0° C. for 20 minutes and then allowed to warm to roomtemperature. Propagation of the reaction was monitored by TLC(EtOAc/Hexane; 1:1) and indicated termination after 45 minutes.

The reaction was quenched with triethylamine, diluted with EtOAc, andextracted with saturated (aq.) NaHCO₃, brine, dried over MgSO₄ andconcentrated. The crude was purified by flash chromatography (silica,EtOAc/Hexane) to yield the neamine moiety (18, FIG. 13), 133.4 mg (99%yield) (full NMR and mass spectra data are given in the reference above)and compound 19 a (FIG. 13), 47 mg (32% yield).

¹H NMR (500 MHz, CDCl₃) data of 19 a: δ=2.12 (s, 3H, OCOMe), 2.16 (s,3H, OCOMe), 2.17 (s, 3H, OCOMe) 2.35 (s, 3H, MeSTol), 3.33 (dd, 1H,J₁=4.5, J₂=13.0 Hz, HII-6), 3.45 (bs, 1H, HII-2), 3.59 (dd, 1H, J₁=7.0,J₂=12.5 Hz, HII-6′), 3.78 (dd, 1H, J₁=2.5, J₂=12.5 Hz, HI-5), 3.85 (dd,1H, J₁=2.5, J₂=12.5 Hz, HI-5′), 4.07 (dd, 1H, J₁=J₂=6.0 Hz, HII-5), 4.20(m, 1H, HI-4), 4.53 (dd, 1H, J₁=J₂=5.5 Hz, HI-3), 4.72 (bs, 1H, HII-4),4.90 (s, 1H, HII-1), 5.45 (bs, 1H, HII-3), 5.20 (dd, 1H, J₁=J₂=3.5 Hz,HI-2′), 5.38 (d, 1H, J=3.0 Hz, HI-1), 7.16 (d, J=7.5 Hz, 2H, ortho tothe methyl of S-Tol), 7.41 (d, J=8.0 Hz, 2H, ortho to the sulfur ofS-Tol).

¹³C NMR: δ=20.7, 20.7, 20.8, 50.7 (CI-6), 56.6 (CII-2), 61.3 (CII-5),65.6 (CII-4), 68.7, 73.2 (CI-2), 75.3 (CI-2), 76.5 (CI-3), 83.1 (CI-4),88.6 (CI-1), 99.3 (CII-1), 128.3, 130.0, 133.1, 138.6, 168.5, 169.8,170.2.

MALDI-TOFMS: m/z=633.1 (M+K⁺, C₂₄H₃₀N₆O₁₀S requires 633.3).

Compound 19 b (FIG. 13) was prepared by deacetylation of compound 19 a,using sodium methoxide in methanol, followed by selective protection ofthe primary alcohol by TBDPS and treatment with acetic anhydride inpyridine.

General procedure for the coupling of donors 5 c, 5 e, 8 b, 19 a andacceptor 1: To a powdered, flame dried 4 Å molecular sieves (0.5 gram)was added CH₂Cl₂ (5 mL), followed by the addition of acceptor 1 (150 mg,0.147 mmol) and donor 5 e (65 mg, 0.293 mmol). After being stirred for10 minutes at room temperature, the mixture was treated with NIS (66.0mg, 0.286 mmol). After additional 5 minutes at room temperature, TfOH(cat) was added. Propagation of the reaction was monitored by TLC (EtOAc50%, Hexane 50%), which indicated completion after 10 minutes. Thereaction was diluted with EtOAc, and filtered through celite. Afterthorough washing of the Celite with EtOAc, the washes were combined andextracted with 10% Na₂S₂O₃, saturated (aq.) NaHCO₃, brine, dried overMgSO₄ and concentrated. The crude was purified by flash chromatographyto yield 16 g (207 mg, 91% yield).

¹H NMR (500 MHz, CDCl₃) data of 16 g are summarized in Table 8hereinbelow.

¹³C NMR: δ=20.3, 20.7, 20.8, 21.0, 31.3 (C-2), 50.3 (C-6′″), 51.2 and51.4 (C6′ and C-6″″), 54.6, 56.7, 58.3, 59.0, 60.6, 65.4, 68.6 (C-5″),68.9, 69.8, 70.3, 70.4, 70.7, 72.7, 73.7, 75.3, 75.5, 77.4, 81.1, 82.3,96.5 (C-1′), 98.3 (C-1″″), 99.6 (C-1′″), 107.4 (C-1), 122.8, 123.1,128.3, 128.4, 128.6, 129.7, 129.8, 129.9, 130.4, 133.3, 133.5, 165.1,165.7, 168.4, 169.6, 169.7, 169.8, 169.9, 170.3, 177.1.

ESIMS: m/z=1585.3 (M+K⁺, C₆₃H₆₆ N₂₂O₂₆ requires 1585.8).

Compound 16 f: The titled compound was prepared as was described for thepreparation of compound 16 g. The conditions used were: Donor 5 c (149mg, 0.306 mmol), acceptor 1 (250 mg, 0.245 mmol), NIS (68 mg, 0.302mmol), TfOH (cat.), acetonitrile anhydrous (7 mL), 4 Å molecular sieves(500 mg) to yield 16 f (271 mg, 80%).

¹H NMR (500 MHz, CDCl₃) data of 16 f are summarized in Table 9hereinbelow.

¹³C NMR: δ=20.7, 20.8, 20.9, 30.2 (C-2), 50.3 and 51.0 (C-6′″), C-6′″could not be distinguished), 53.6 (C-5″″), 56.3, 58.1, 58.8, 60.9, 64.6,65.4, 67.3 (C-5″), 68.6, 69.2, 69.4, 70.1, 72.6, 73.3, 74.2, 75.4, 75.4,80.1, 80.3, 81.3, 96.3 (C-1′), 98.9 (C-1′″), 105.6 (C-1″), 107.3(C-1′″″), 124.8, 128.4, 128.5, 128.7. 129.1, 129.7, 129.7, 133.5, 133.6,165.3, 165.5, 168.6, 169.4, 169.7, 169.8, 170.2, 170.3.

ESIMS: m/z=1426.4 (M+K⁺, C₅₄H₆₁N₂₁O₂₄ requires 1426.2).

Compound 16 e: The titled compound was prepared as described for thepreparation of compound 16 g. The conditions used were: Donor 8 b (207mg, 0.36 mmol), acceptor 1 (250 mg, 0.24 mmol), NIS (81 mg, 0.36 mmol),TfOH (cat.), acetonitrile anhydrous (8 mL), 4 Å molecular sieves (800mg), −40° C., to yield 16 e (226 mg, 87%).

¹H NMR (500 MHz, CDCl₃) data of 16 e are summarized in Table 10hereinbelow.

¹³C NMR: δ=20.5, 20.7, 20.8, 31.0 (C-2), 49.9 (C-6′″), 51.1 (C-6′),56.2, 58.1, 58.9, 60.5, 60.8 (C-5″), 65.2, 67.7 (C-6″″), 68.4, 68.5,69.4, 70.4, 71.9, 72.3, 72.6, 73.6, 74.7, 74.9, 75.5, 76.6, 80.2, 83.4,96.6 (C-1), 98.7 (C-1′″), 101.1 (C-1″″), 108.2 (C-1″), 128.5, 128.7,129.5, 129.7, 129.8, 133.1, 133.5, 164.9, 169.6, 169.7, 169.9, 170.2,170.7.

ESIMS: m/z=1498.3 (M+K⁺, C₅₇H₆₄N₂₁O₂₆ requires 1497.8).

Compound 16 h: The titled compound was prepared as described for thepreparation of compound 16 g. The conditions used were: Donor 19 a (135mg, 0.212 mmol), acceptor 1 (150 mg, 0.147 mmol), NIS (52 mg, 0.231mmol), TfOH (cat.), acetonitrile anhydrous (5 mL), 4 Å molecular sieves(600 mg), to yield 16 h (101.7 mg, 67%).

¹H NMR (500 MHz, CDCl₃) data of 16 h are summarized in Table 11hereinbelow.

¹³C NMR: δ=20.5, 20.6, 20.7, 20.8, 31.4 (C-2), 50.5 and 50.6 (C-6′″″ andC-6′″), 51.0 (C-6′), 56.1, 56.4, 58.0, 59.0, 60.6, 64.4 (C-5″″), 65.6,65.7, 68.6 (C-5″), 68.7, 68.8, 69.3, 69.3, 69.9, 73.4, 73.9, 74.1, 74.2,75.3, 75.4, 76.2, 76.3, 78.8, 79.9, 81.1, 96.3 (C-1′), 98.6 (C-1′″″),98.8 (C-1′″), 105.1 (C-1″″), 106.8 (C-1″), 168.5, 168.6, 169.5, 169.6,169.8, 169.8, 169.9, 170.0, 170.2, 170.8.

MALDI-TOFMS: m/z=1574.3 (M+K⁺, C₅₄H₇₀N₂₄O₃₀ requires 1574.3).

Compound 16 j: The titled compound was prepared as was described for thepreparation of compound 16 g. The conditions used were: Donor 5 a (150mg, 0.372 mmol), acceptor 1 (250 mg, 0.245 mmol), NIS (104.6 mg, 0.465mmol), TfOH (cat.). Acetonitrile (8 mL), 4 Å molecular sieves (800 mg),−40° C., to yield 16 j (282 mg, 85%).

¹H NMR (500 MHz, CDCl₃) data of 16 j are summarized in Table 16hereinbelow.

¹³C NMR (125 MHz, CDCl₃): δ=20.4, 20.5, 20.6, 20.7, 20.8, 31.7 (C-2),50.8 (C-6′″), 52.4 (C-6′), 57.3, 58.3, 59.0, 60.5, 61.2 (C-5″), 65.2,68.1 (C-6″″), 68.4, 68.5, 69.4, 71.1, 71.7, 72.3, 72.6, 73.8, 74.5,75.1, 75.8, 76.6, 80.8, 83.4, 97.1 (C-1), 98.1 (C-1′″), 101.4 (C-1″″),110.2 (C-1″), 163.9, 164.2, 166.9, 169.1, 169.4, 169.6, 169.7, 169.9,170.2, 170.7.

MALDI-TOFMS m/z 1392.4 (M+K⁺, C₄₉H₆₄N₁₈O₂₈ requires 1392.1).

The preparation of compounds II-XIII according to the present inventionis now described.

Compound II was prepared as follows: Compound 16 a (0.11 gram, 0.078mmol) was dissolved in 33% solution of MeNH₂ in EtOH (40 mL) and themixture was stirred at room temperature for 30 hours. The reagent andthe solvent were removed by evaporation and the residue was dissolved inTHF (10 mL), NaOH 0.1M (2 mL) and stirred at 60° C. for 10 minutes afterwhich PMe₃ (1 M solution in THF, 3.73 mL, 3.73 mmol) was added.Propagation of the reaction was monitored by TLC (CH₂Cl₂/MeOH/H₂O/MeNH₂,33% solution in EtOH, 10:15:6:15, Rƒ=0.33), which indicated completionafter 3.5 hours. The reaction mixture was purified by flashchromatography on a short column of silica and the column was washed asfollows: THF, EtOAc, MeOH/EtOAc (1:1), MeOH, and finally with MeNH₂ (33%solution in EtOH). The fractions containing the product were evaporatedunder vacuum, re-dissolved in water and evaporated again to afford theproduct as a free amine (48.7 mg, 81%). This product was then dissolvedin water, the pH was adjusted to 7.5 with 0.01 M H₂SO₄ and the solutionwas then lyophilized to give the sulfate salt of Compound II (88.5 mg)as a white foamy solid.

¹H NMR (500 MHz, D₂O, pH 4.5, sulfate salt): δ=1.85 (ddd, 1H,J₁=J₂=J₃=12.5 Hz, H-2 axial), 2.38 (dt, 1H, J₁=4.5 J₂=12.5, H-2equatorial), 3.07-4.53 (m, 26H), 4.78 (d, 1H, J=7.0 Hz, anomericproton), 5.21 (d, 1H, J=1.5 Hz, anomeric proton), 5.34 (d, 1H, J=3.0 Hz,anomeric proton), 5.97 (d, 1H, J=4.0 Hz, anomeric proton).

¹³C NMR (125.8 MHz, D₂O, pH 4.5, sulfate salt): δ=29.8, 34.6, 42.2,42.3, 49.9, 50.3, 51.2, 51.6, 52.6, 55.1, 62.3, 64.3, 69.2, 69.4, 69.5,69.9, 71.3, 71.9, 72.8, 73.8, 74.1, 74.9, 77.1, 82.0, 86.8, 96.9(anomeric carbon), 97.1 (anomeric carbon), 102.0 (anomeric carbon),112.1 (anomeric carbon).

ESIMS: m/z=781.2(M+Li⁺, C₂₉H₅₈N₈O₁₆ requires 781.2).

Compound III was prepared as was described for the preparation ofCompound II with the following quantities: the product (β-anomer) thatwas obtained by the partial deprotection of compound 16 b (122.4 mg,0.124 mmol), THF (9 mL), NaOH 0.1M (3 mL), PMe₃ (1M solution in THF, 6.8mL, 6.8 mmol), gave the product as a free amine (93.1 mg, 96%). Thisproduct was dissolved in water, the pH was adjusted to 7.5 with 0.01 MH₂SO₄, and the solution was lyophilized to afford the sulfate salt ofCompound III (134.5 mg) as a white foamy solid.

¹H NMR (500 MHz, D₂O, pH 4.5, sulfate salt): δ=1.77 (ddd, 1H,J₁=J₂=J₃=12.5 Hz, H-2 axial), 2.38 (bd, 1H, H-2 equatorial), 2.83-4.39(m, 26H), 4.54 (d, 1H, J=7.5 Hz, anomeric proton), 5.17 (s, 1H, anomericproton), 5.31 (s, 1H, anomeric proton), 5.98 (s, 1H, anomeric proton).

¹³C NMR (125.8 MHz, D₂O, pH 4.5, sulfate salt): δ=20.8, 23.6, 30.0,42.2, 42.3, 42.5, 50.2, 51.8, 52.7, 55.1, 55.2, 55.8, 69.3, 69.4, 69.6,69.9, 71.1, 71.6, 72.0, 73.7, 74.3, 74.4, 75.0, 77.1, 81.3, 86.9, 96.4(anomeric carbon), 97.0 (anomeric carbon), 105.1 (anomeric carbon),112.5 (anomeric carbon).

ESIMS: m/z=813.2 (M+K⁺, C₂₉H₅₈N₈O₁₆ requires 813.8).

Compound IV was prepared as was described for the preparation ofCompound II with the following quantities: 16 c (180 mg, 0.113 mmol) wastreated in the first step with 33% solution of MeNH₂ in EtOH (40 mL) for40 hours; the product from this step was dissolved in THF (10 mL), NaOH0.1M (2 mL), and treated with PMe₃ (1M solution in THF, 4.05 mL, 4.05mmol) to yield Compound IV as a free amine (73.1 mg, 84%). The amine wasdissolved in water, the pH was adjusted to 7.5 with H₂SO₄ (0.01 M), andthe solution lyophilized to give the sulfate salt of Compound IV (117mg) as a white foamy solid.

¹H NMR (500 MHz, D₂O, pH 3.75, sulfate salt): δ=1.94 (ddd, 1H,J₁=J₂=J₃=12.5 Hz, H-2 axial), 2.35 (broad dt, H-2 equatorial), 2.96 (t,1H, J=10.0 Hz, H-2″″), 2.97 (broad t, 1H, H-3′″), 3.05 (dd, 1H, J₁=8.5J₂=13.0 Hz), 3.21-3.33 (m, 5H, H-4″″), 3.37-3.47 (m, 5H, H-2′, H-2′″),H-5″″), 3.61-3.70 (m, 4H, H-4′″), H-3″″), 3.76-3.80 (m, 2H), 3.83-3.90(m, 2H), 3.96 (t, 1H, J=10.0 Hz, H-3′), 4.14-4.20 (m, 2H, H-5″), 4.23(broad t, 1H), 4.31 (broad t, 1H, H-3″), 4.41 (broad t, 1H, H-2″), 4.79(d, J=8.5 Hz , 1H, H-1′″), 5.19 (s, 1H, H-1′″), 5.32 (s, 1H, H-1″), 5.98(d, J=4.0 Hz, 1H, H-1′).

¹³C NMR: δ=26.4, 29.6, 42.2, 42.5, 50.3, 51.6, 52.6, 55.1, 57.4, 61.8,68.9, 69.4, 69.7, 70.8, 71.4, 71.6, 72.3, 73.2, 73.6, 74.0, 74.6, 76.2,76.6, 77.9, 81.7, 87.3, 96.70 (anomeric carbon), 96.73 (anomericcarbon), 101.0 (anomeric carbon), 112.5 (anomeric carbon).

ESIMS: m/z=798.2 (M+Na⁺, C₂₉H₅₇N₇O₁₇ requires 798.3).

Compound V was prepared as follows: A catalytic amount of NaOMe (0.5Msolution in MeOH) was added to a suspension of compound 16 d (140 mg,0.11 mmol) in dry MeOH (10 mL) at 0° C., and the propagation of thereaction was monitored by TLC (MeOH 10%, dichloromethane 90%). After 3hours the mixture was neutralized with Dowex H⁺ and evaporated todryness. The resulted crude was then treated as described for thepreparation of Compound II to yield the free amine of Compound V (57.1mg, 70%).

¹H NMR (500 MHz, D₂O, pH 4.5, sulfate salt): δ=1.86 (ddd, 1H,J1=J2=J3=12.5 Hz, H-2 axial), 2.33(broad dt, H-2 equatorial), 3.10 (dd,1H, J1=7.0 J2=13.0 Hz), 3.20-4.43 (m, 17H), 3.84 (s, 1H, anomericproton), 5.16 (s, 1H, anomeric proton), 5.30 (s, 1H, anomeric proton),5.93 (d, J=3.0 Hz, 1H, anomeric proton).

¹³C NMR (125.8 MHz, D₂O, pH 4.5, sulfate salt): δ=30.2, 42.2, 42.2,50.3, 51.8, 52.6, 55.5, 62.0, 69.0, 69.5, 69.9, 71.2, 72.1, 74.3, 75.1,76.6, 77.1, 83.0, 83.6, 86.7 (anomeric carbon), 96.9 (anomeric carbon),96.9 (anomeric carbon), 112.0 (anomeric carbon).

ESIMS: m/z=769.2 (M+Na⁺, C₂₈H₅₄N₆O₁₇ requires 769.3).

Compound VIII: The titled compound was prepared as was described for thepreparation of Compound II with the following quantities: 16 g (207 mg,0.134 mmol), in 33% solution of MeNH₂ in EtOH (40 mL), THF (4.5 mL),NaOH 0.1M (1 mL), H₂O (1 mL), PMe₃ (1M solution in THF, (2.68 mL, 2.68mmol), to yield of the free amine (81.9 mg, 79%). The product wasdissolved in water and the pH was adjusted to 6.8 by H₂SO₄ (0.01 M), andlyophilized to afford the sulfate salt of Compound VIII as a white foamysolid.

NMR analyses were performed at 500 MHz, in D₂O, and at pH 3.45, adjustedby H₂SO₄).

¹H NMR: δ=1.93 (ddd, 1H, J₁=J₂=J₃=12.5 Hz, H-2 axial) 2.35 (broad dt,1H, H-2 equatorial), 2.98 (t, J=9.0 Hz 1H), 3.05-3.09 (m, 2H), 3.17-3.89(m, 17H), 3.97 (t, J=9.5 Hz 1H), 4.13-4.16 (m, 2H), 4.23-4.30 (m, 3H),4.41 (bs, 1H), 4.56 (dd, 1H, J₁=J₂7.5 Hz), 4.89 (d, 1H, J=8.5 Hz,H-1″″), 5.20 (s, 1H, H-1′″), 5.35 (s, 1H, H-1″), 6.00 (d, 1H, J=4.0 Hz,H-1′).

¹³C NMR: δ=29.8 (C-2), 42.2 (C-6′″) and C-6″″), 42.5 (C-6′), 50.2, 51.7,52.6, 55.2, 57.4, 69.0, 69.4, 69.8, 70.5, 71.2, 72.3, 73.3, 73.6, 73.8,74.1, 74.2, 74.5, 75.9, 76.6, 81.4, 87.1, 96.6, 96.7, 101.4, 112.4.

ESIMS: m/z=813.3 (M+K⁺, C₂₉H₅₈N₈O₁₆ requires 813.3).

Compound VI: The titled compound was prepared as was described for thepreparation of Compound II with the following quantities: compound 16 e(261.2 mg, 0.188 mmol), in 33% solution of MeNH₂ in EtOH (40 mL), THF (6mL), NaOH 0.1M (3 mL), H₂O (2 mL), PMe₃ (1M solution in THF, (10.43 mL,10.43 mmol), to yield the free amine (79.4 mg, 57%). The product wasdissolved in water and the pH was adjusted to 6.8 by H₂SO₄ (0.01 M), andthe solution lyophilized to afford the sulfate salt of Compound VI as awhite foamy solid.

NMR analyses were performed at 500 MHz, in D₂O, and at pH 4.49, adjustedby H₂SO₄).

¹H NMR: δ=1.88 (ddd, 1H, J₁=J₂=J₃=12.5 Hz, H-2 axial) 2.27 (broad dt,1H, H-2 equatorial), 2.90-2.95 (m, 1H), 3.03 (dd, 1H, J₁=8.5, J₂=13.5Hz), 3.03 (dd, 1H, J₁=3.5, J₂=10.5 Hz), 2.20-2.29 (m, 4H), 3.32-3.37 (m,2H), 3.44 (bs, 1H), 3.58 (t, 1H, J=9.5 Hz), 3.66 (dd, 1H, J₁=6.0,J₂=11.0 Hz) 3.79-4.19 (m, 10H), 4.37 (d, 1H, J=4.0 Hz), 4.45 (dd, 1H,J₁=4.5, J₂=7.5 Hz), 4.99 (s, 1H, H-1′″), 5.14 (s, 1H, H-1″″), 5.27 (s,1H, H-1″), 5.97 (d, 1H, J=4.0 Hz, H-1′).

¹³C NMR: δ=27.5 (C-2), 42.3 (C-6′″), 42.4 (C-6′), 44.8 (C-5″″), 50.3,51.7, 52.6, 55.5, 68.7, 69.1 (C-5″), 69.4, 69.8, 71.2, 72.3, 73.2, 74.0,74.1, 74.2, 75.8, 75.9, 76.9, 87.2, 96.4 (C-1′″) and C-1′), 109.7(C-1″″), 112.4 (C-1″).

ESIMS: m/z=784.4 (M+K⁺, C₂₈H₅₅N₇O₁₆requires 784.8).

Compound VII: The titled compound was prepared as was described for thepreparation of Compound II with the following quantities: compound 16 f(220 mg, 0.151 mmol), in 33% solution of MeNH₂ in EtOH (60 mL), THF (4.5mL), NaOH 0.1M (1 mL), H₂O (1 mL), PMe₃ (1M solution in THF, (2.64 mL,2.64 mmol), to yield of the free amine (98.3 mg, 84%). The product wasdissolved in water and the pH was adjusted to 6.8 by H₂SO₄ (0.01 M), andthe solution was lyophilized to afford the sulfate salt of Compound IVas a white foamy solid.

NMR analyses were performed at 500 MHz, in D₂O, and at pH 4.2, adjustedby H₂SO₄).

¹H NMR: δ=1.92 (ddd, 1H, J₁=J₂=J₃=12.5 Hz, H-2 axial) 2.32 (broad dt,1H, H-2 equatorial), 2.98-3.05 (m, 2H), 3.20-3.47 (m, 10H), 3.56-3.86(m, 10H), 3.92 (t, J=10.0 Hz 1H), 4.09-4.20 (m, 5H), 4.38 (d, 1H, J=3.5Hz), 4.46 (d, 1H, J=8.0 Hz, H-1″″), 5.16 (s, 1H, H-1′″), 5.29 (s, 1H,H-1 ″), 5.96 (d, 1H, J=3.5 Hz, H-1′).

¹³C NMR: δ=29.7 (C-2), 42.2 (C-6′″), 42.6 (C-6′), 50.2, 51.7, 52.6,54.1, 55.1, 62.0 (C-6″″), 69.2 (C-5″), 69.4, 69.8, 71.3, 72.0, 73.3,73.7, 74.0, 74.2, 74.4, 75.2, 75.3, 76.9, 81.5, 87.0, 96.4 (C-1′″), 97.0(C-1′), 104.8 (C-1″″), 112.6 (C-1″).

ESIMS m/z: 814.3 (M+K⁺, C₂₉H₅₇N₇O₁₇requires 814.3).

Compound X: The titled compound was prepared as was described for thepreparation of Compound II with the following quantities: compound 16 h(101.7 mg, 0.066 mmol), in 33% solution of MeNH₂ in EtOH (40 mL), THF(4.5 mL), NaOH 0.1M (0.5 mL), H₂O (0.5 mL), PMe₃ (1M solution in THF,(1.6 mL, 1.6 mmol), to yield of the free amine (48.2 mg, 80%). Theproduct was dissolved in water and the pH was adjusted to 6.8 by H₂SO₄(0.01 M), and the solution was lyophilized to afford the sulfate salt ofCompound X as a white foamy solid.

NMR analyses were performed at 500 MHz, in D₂O, and at pH 3.45, adjustedby H₂SO₄).

¹H NMR: δ=1.93 (ddd, 1H, J₁=J₂=J₃=12.5 Hz, H-2 axial) 2.34 (broad dt,1H, H-2 equatorial), 3.06-4.39 (m, 33H), 5.00 (d, 1H, J=2.5 Hz, H-1″″),5.17 (s, 2H, C-1′″, C-1′″″), 5.31 (d, 1H, J=2.5 Hz, H-1″), 5.98 (d, 1H,J=3.5 Hz, H-1′).

¹³C NMR: δ=29.6 (C-2), 42.2 and 42.3 (C-6′, C-6′″, C6′″″ could not bedistinguished), 50.6, 51.6, 52.6, 52.7, 55.4, 63.3 (C-5″″), 69.0, 69.1,69.4, 69.7, 70.5 (C-5″), 71.3, 72.0, 72.1, 72.9, 74.0, 74.7, 74.9, 76.5,77.7, 79.0, 82.0, 80.1, 84.2, 87.0, 96.4 (C-1′), 97.0 and 97.5 (C-1′″),C-1′″″ could not be distinguished) 109.0 (C-1″″), 112.4 (C-1″).

MALDI-TOFMS: m/z=945.5 (M+K⁺, C₃₄H₆₆N₈O₂₀requires 945.9).

Compound XI: Compound 16 i (0.164 gram, 0.11 mmol) was dissolved in 33%solution of MeNH₂ in EtOH (40 mL) and the mixture was stirred at roomtemperature for 24 hours. The reagent and the solvent were removed byevaporation and the residue was dissolved in THF (4 mL), NaOH 0.1M (1mL) and stirred at room temperature for 10 minutes after which PMe₃ (1Msolution in THF, 1.66 mL, 1.66 mmol) was added. Propagation of thereaction was monitored by TLC (CH₂Cl₂/MeOH/H₂O/MeNH₂, 33% solution inEtOH; 10:15:6:15), which indicated completion after 4.5 hours. Thereaction mixture was purified by flash chromatography on a short columnof silica and the column was washed as follows: THF, EtOAc, MeOH/EtOAc(1:1), MeOH, and finally the product was eluted with MeNH₂ (33% solutionin EtOH). The fractions containing the product were evaporated undervacuum, re-dissolved in water and evaporated again to afford the freeamine compound (61.2 mg, 73%). The product was then dissolved in water,and the pH was adjusted to 6.8 by H₂SO₄ (0.01 M), and the product waslyophilized to give the sulfate salt of Compound XI as a white foamysolid.

¹H NMR (500 MHz, D₂O, pH=3.04) data of XI are summarized in Table 15hereinbelow.

¹³C NMR: δ=29.6 (C-2), 35.2 (C-5″″), 42.2 (C-6′), 42.3 (C-6′″), 50.3,51.6, 52.6, 55.3, 63.5 (C-5″), 69.0, 69.4, 69.7, 71.3, 72.1, 72.7, 73.0,74.1, 75.0, 76.2, 76.4, 79.8, 82.1, 87.1, 87.2, 89.4 (C-1″″), 96.2(C-1′″), 97.1 (C-1′), 112.5 (C-1″).

MALDI-TOFMS: m/z=801.1 (M+K⁺, C₂₈H₅₄N₆O₁₆S requires 801.3).

Compound XII: The titled compound was prepared as was described for thepreparation of Compound XI with the following quantities: 16 j (282 mg,0.208 mmol), in 33% solution of MeNH₂ in EtOH (40 mL), THF (4.5 mL),NaOH 0.1M (1 mL), H₂O (1 mL), PMe₃ (1M solution in THF, (1.87 mL, 1.87mmol), to yield of the free amine: 111.5 mg, (69%). The product wasdissolved in water and the pH was adjusted to 6.8 by H₂SO₄ (0.01 M), andlyophilized to afford the sulfate salt of XII as a white foamy solid.

¹H NMR (500 MHz, D₂O, pH 4.2, adjusted by H₂SO₄ 0.01M) data of CompoundXII are summarized in the attached Table 9.

¹³C NMR (125 MHz, D₂O): δ=29.6 (C-2), 42.2 (C-6′″), 42.7 (C-6′), 50.1,51.7, 52.6, 55.0, 62.3 (C-5″), 69.2 (C-6″″), 69.3, 69.4, 69.6, 71.3,71.5, 72.0, 73.5, 74.1, 74.4, 74.8, 75.0, 76.7, 77.4, 77.6, 81.4, 87.0,96.4 (C-1′), 97.0 (C-1′″), 104.9 (C-1″″), 112.6 (C-1″″).

MALDI-TOFMS: m/z=815.2 (M+K⁺, C₂₉H₅₆N₆O₁₆requires 815.7).

Compounds IX and XIII: Lewis acid (BF₃.Et₂O) promoted coupling of thethiol acceptor 2 with the trichloroacetimidate donor 8 e furnished thecorresponding protected β-thioglycoside, which after two-stepsdeprotection, provided the designed thioglycoside 16 i in 73% isolatedyield. When the chromatographically pure thioacetate 2 a was directlysubjected to the same two-steps deprotection procedure, treatment withmethylamine followed by Staudinger reaction, a mixture (a ratio of about1:3) of Compound IX and the corresponding disulfide dimer Compound XIIIwere obtained in an overall yield of 88%. This mixture was purified on aBiogel P-2 column to yield the sufficiently pure Compounds IX and XIIIfor biological tests.

Compound 2 a (250 mg, 0.231 mmol) was dissolved in 33% solution of MeNH₂in EtOH (40 mL) and the mixture was stirred at room temperature for 24hours. The reagent and the solvent were removed by evaporation and theresidue was dissolved in THF (4 mL), NaOH 0.1M (1 mL) and stirred at 0°C. After 10 minutes, PMe₃ (1M solution in THF, 1.66 mL, 1.66 mmol) wasadded. Propagation of the reaction was monitored by TLC(CH₂Cl₂/MeOH/H₂O: MeNH₂ (33% solution in EtOH); 10:15:6:15), whichindicated the full consumption of the starting material after 2.5 hours.At this point two products appeared on the TLC. The polar dimmer productappeared with the R_(ƒ) value of 0.16 and the less polar monomerappeared with the R_(ƒ) value of 0.54. The reaction mixture was purifiedby flash chromatography on a short column of silica gel and the columnwas washed as follows: THF, EtOAc, MeOH/EtOAc (1:1), MeOH, and finallythe product was eluted with MeNH₂ (33% solution in EtOH). The fractionscontaining the mixture of monomer and dimmer products were evaporatedunder vacuum, re-dissolved in water and evaporated again to afford themixture of free amines (128.3 mg, 88%). The mixture was then dissolvedin water, the pH was adjusted to 6.8 by H₂SO₄ (0.01 M), and lyophilizedto give the sulfate salts of the amines as a white foamy solid. Themonomer and dimer were then separated using size exclusionchromatography (P-2 gel was packed in a column 47 cm length, 1.2 cmdiameter column). The pure dimer Compound XIII and monomer Compound IXwere then lyophilized.

Following are the data obtained in NMR and MS measurements of the purethiol, Compound IX.

¹H NMR (500 MHz, D₂O pD=6.8): δ=1.45 (ddd, 1H, J₁=J₂=J₃=12.5 Hz, H2-ax),2.08 (bd, 1H, J=12.0 Hz, H2-eq), 2.54 (dd, 1H, J₁=3.0, J₂=13.0 Hz, H5″),2.66 (dd, 1H, J₁=9.0, J₂=13.0 Hz, H5″), 2.90-3.13 (m, 5H,), 3.20-3.33(m, 4H), 3.38 (bs, 1H), 3.47 (bt, 1H, J=9.0 Hz), 3.58 (bt, 1H, J=9.0Hz), 3.64-3.75 (m, 3H), 3.87 (m, 1H), 4.06 (bs, 1H), 4.17 (bs, 1H),4.27-4.37 (m, 3H), 5.11 (bs, 1H, H1′), 5.23 (bs, 1H, H1′″), 5.78 (bd,1H, J=3.5 Hz, H1″).

¹³C NMR (125 MHz, D₂O): δ=40.5, 40.7, 49.3, 50.6, 51.3, 54.3, 64.6,68.0, 68.3, 69.2, 70.5, 71.2, 73.6, 73.7, 77.9, 80.1, 85.8, 96.6, 96.8,110.6.

MALDI-TOF MS m/z 669.2(M+K⁺, C₂₈H₅₄N₆O₁₆S requires 669.5).

Following are the data obtained in NMR and MS measurements of the purethiol, Compound XIII.

¹H NMR (500 MHz, D₂O pD=6.8): δ=1.82 (m, 2H, H2axial), 2.26 (bd, J=12.0Hz, 2H, H₂ equatorial), 2.80 (dd, 1H, J₁=8.5, J₂=13.0 Hz, H5″), 3.08(dd, 1H, J₁=8.0, J₂=13.0 Hz, H5″), 3.13-3.36 (m, 12H), 3.60 (bt, 2H,J=10.0 Hz), 3.68 (bs, 2H), 3.83-3.92 (m, 2H), 3.99 (bt, 2H, J=9.5 Hz),4.10 (bs, 2H), 4.19 (bs, 2H), 4.36-4.40 (m, 6H), 5.18 (bs, 2H, H1′),5.33 (bs, 2H, H1′″), 5.93 (bd, 2H, J=3.0 Hz, H1′).

¹³C NMR (125 MHz, D₂O ): δ=40.3, 48.5, 49.8, 50.7, 53.6, 67.2, 67.6,68.4, 69.2, 70.3, 70.9, 72.4, 73.2, 75.5, 78.4, 78.9, 85.3, 94.4(C-1′),95.3 (C-1′″), 110.1 (C-1″).

MALDI-TOF MS m/z 1298.5 (M+K⁺, C₂₈H₅₄N₆O₁₆S requires 1298.2).

NMR Methods and Results

¹H NMR, ¹³C NMR, DEPT, COSY, 2D TOCSY, 1D TOCSY, HMQC, HMBC spectra wererecorded on a Bruker Advance 500 spectrometer, and chemical shiftsreported (in ppm) are relative to internal Me₄Si (δ=0.0) with CDCl₃ asthe solvent, and to HOD (δ=4.63) with D₂O as the solvent. Massspectrometric analysis was performed using a Bruker Daltonix Apex 3 massspectrometer using electron spray ionization (ESI), using a TSQ-70B massspectrometer (Finnigan-MAT) or using matrix assisted laser desorptionionization with a time of flight mass detector (MALDI-TOF by Micromass)using α-cyano-4-hydrocinnamic acid as a matrix. Reactions were monitoredby TLC on Silica Gel 60 F₂₅₄ (0.25 mm, Merck), and spots were visualizedby charring with a yellow solution containing (NH₄)Mo₇O₂₄4H₂O (120grams) and (NH₄)₂Ce(NO₃)₆ (5 grams) in 10% H₂SO₄ (800 mL). Flash columnchromatography was performed on Silica Gel 60 (70-230 mesh). Allreactions were carried out under an argon atmosphere with anhydroussolvents, unless otherwise noted. All chemicals unless otherwise stated,were obtained from commercial sources.

Chemical structures corresponding to the previously described compounds,followed by the NMR results for those structures, are given below. TABLE1

¹H NMR (500 MHz, CDCl₃) chemical shifts and coupling constants for thetitled structure.^(a) Ring H1 H2 H3 H4 H5 H5′ H6 H6′ A 6.09 3.14 5.454.92 4.42 3.25-3.32 3.25-3.32 d dd t t ddd m m J = 3.5 J = 3.5, J = 9.5J = 10.0 J = 3.0, 10.5 5.0, 8.5 C 5.36 4.77 4.36 4.23 3.71 3.86 d t t dddd m J = 4.5 J = 4.5 J = 4.7 J = 4.0, J = 4.5, 6.0 11.5 D 4.79 3.25-3.325.00 4.67 3.49 3.25-3.32 3.54 d m t s ddd. m dd J = 2.0 J = 3.0 J = 2.0,J = 8.5, 4.5, 6.5 13.0 H1 H2eq H2ax H3 H4 H5 H6 B 4.92 1.58 2.34 3.493.67 3.86 3.39 t ddd dt ddd t m ddd J = 1.0 J₁ = J₂ = J₃ J = 4.0, J₁ =J₂ = J = 9.0 J = 4.0, = 12.5 13.5 J₃ = 5.0 10.0, 12.5^(a)Values of chemical shifts are in ppm and values of couplingconstants. are in Hz. The additional peaks in the spectrum wereidentified as follow: δ 0.06 (s, 3H, Me), 0.08 (s, 3H, Me), 1.07 (s, 9H,t-Bu), 2.01 (s, 3H, acetate), 2.03 (s, 3H, acetate), 2.06 (s, 3H,acetate), 2.12 (s, 3H, acetate), 2.14 (s, 3H, acetate), 2.16 (s, 3H,acetate).

TABLE 2

¹H NMR (500 MHz, CDCl₃) chemical shifts and coupling constants for theneomycin acceptor 1.^(a) Ring H1 H2 H3 H4 H5 H5′ H6 H6′ A 6.02 3.21 5.615.08-5.15 4.60 3.47 3.40-3.42 d dd dd m ddd dd m J = 4.0 J = 3.5, J =9.5, J = 3.0, J = 2.5, 10.5 10.5 5.5, 10.0 13.5 C 5.46 4.93 4.514.19-4.24 4.02 3.83 d dd t m dd dd J = 2.5 J = 3.0, J = 6.5 J = 4.0, J =6.0, 5.5 12.5 12.5 D 4.99 3.40-3.42 5.08-5.15 4.80 4.19-4.24 3.703.40-3.42 d m m s m dd m J = 1.5 J = 8.5, 13.0 H1 H2eq H2ax H3 H4 H5 H6B 3.57 2.51 1.74 3.65 3.82 4.06 5.08-5.15 ddd dt ddd ddd t t m J = 4.5,J = 4.5, J₁ = J₂ = J₃ J = 4.5, J = 9.0 J = 9.0 10.5, 14.5 13.0 = 12.510.0, 14.0^(a)Values of chemical shifts are in ppm and values of couplingconstants are in Hz. The additional peaks in the spectrum wereidentified as follow: δ 1.85 (broad s, 1H, OH), 2.17 (s, 3H, acetate),2.20 (s, 3H, acetate), 2.22 (s, 3H, acetate), 2.25 (s, 3H, acetate),2.27 (s, 3H, acetate), 2.28 (s, 3H, acetate).

TABLE 3

¹H NMR (500 MHz, CDCl₃) chemical shifts and coupling constants for theprotected pseudo-pentasaccharide 16a.^(a) Ring H1 H2 H3 H4 H5 H5′ H6 H6′A 6.12 3.24-3.30 5.42 5.02 4.46-4.52 3.34-3.42 3.63-3.67 d m t t m m m J= 3.5 J = 10 J = 11.5 C 5.16 4.70 4.32 4.08 3.71 3.86 d d dd m dd bd J =4.5 J = 5 J = 6.5, J = 4.5, 11.5 11.5 D 4.46-4.52 3.12 4.96 4.643.63-3.67 3.34-3.42 3.52-3.56 m bs s s m m m E 4.86 5.28 4.46-4.52 4.914.96 4.46-4.52 4.40 d dd m bdd dd m dd J = 8.0 J = 3.0, J₁ = 2.0, J =4.5, 7.5 12.5 12.5 H1 H2eq H2ax H3 H4 H5 H6 B 3.34-3.42 2.31 1.553.24-3.30 4.73 3.75 3.69 m m ddd m t t t J₁ = J₂ = J₃ J = 11.0 J = 9.5 J= 9.0 = 12.5aValues of chemical shifts are in ppm and values of coupling constantsare in Hz. The additional peaks in the spectrum were identified asfollow: δ 2.04 (s, 3H, acetate), 2.05 (s, 3H, acetate), 2.06 (s, 3H,acetate), 2.08 (s, 3H, acetate), 2.14 (s, 3H, acetate), 2.15 (s, 3H,acetate) 4.23 (s, 2H, chloroacetate), 7.57 (t, 2H, meta benzoylprotons), 7.65(t, 1H, para benzoyl proton), 8.06 (d, 2H, ortho benzoylprotons).

TABLE 4

¹H NMR (500 MHz, CDCl₃) chemical shifts and coupling constants for thetitled structure.^(a) Ring H1 H2 H3 H4 H5 H5′ H6 H6′ A 5.77 3.22-3.293.77 3.22-3.29 3.98 3.49 3.40 d m t m ddd dd dd J = 4.0 J = 9.0 J = 3.0,J = 2.5, J = 4.5, 6.0, 9.5 9.5 13.0 C 5.18 4.04 4.24 4.13-4.14 3.43-3.493.43-3.49 d dd dd m m m J = 4 J₁ = J₂ = J₁ = J₂ = 4.0 4.5 D 4.98 3.683.87 3.21-3.25 3.90-3.92 3.54-3.59 3.21-3.29 d bs s m m m m J = 1.0 E4.21 3.24-3.3 3.46-3.49 3.24-3.3 3.36-3.39 3.36-3.39 3.36-3.39 d m m m mm m J = 8.5 H1 H2eq H2ax H3 H4 H5 H6 B 3.24-3.36 2.13 1.55 3.24-3.363.24-3.36 3.54-3.57 3.48 m dt ddd m m m t J = 4.5, J₁ = J₂ = J₃ J = 8.513.0 = 12.5^(a)Values of chemical shifts are in ppm and values of couplingconstants are in Hz.

TABLE 5

¹H NMR (500 MHz, CDCl₃) chemical shifts and coupling constants for thetitled structure.^(a) Ring H1 H2 H3 H4 H5 H5′ H6 H6′ A 5.73 3.09-3.133.77 3.26-3.3 3.98 3.47 3.39 d m t m ddd bd dd J = 4.0 J = 8.5 J = 2.0,J = 5.5, 5.5, 8.5 14.5 C 5.36 4.77 4.36 4.23 3.71 3.86 d t t dd dd m J =4.5 J = 4.5 J = 4.7 J = 4.0, J = 4.5, 6.0 11.5 D 4.99 3.66 3.86 3.273.41-3.52 3.41-3.52 3.41-3.52 d bs bs bs m m m J = 1.0 E 5.86 4.85 4.853.33-3.39 3.42-3.46 3.33-3.39 3.23 d dd dd m m m dd J = 5.5 J₁ = J₂ = J= 5.2, 5.3 J = 3.0, 4.5 17.0 H1 H2eq H2ax H3 H4 H5 H6 B 3.26-3.36 2.121.31 3.26-3.36 3.51-3.61 3.51-3.61 3.41 m dt ddd m m m. t J = 4.0, J₁ =J₂ = J₃ J = 9.0 13.0 = 12.5^(a)Values of chemical shifts are in ppm and values of couplingconstants are in Hz. The additional peaks in the spectrum wereidentified as follow: δ 7.2-7.32 (m, 3H, benzoyl), 7.53-7.55(m, 2H,benzoyl).

TABLE 6

¹H NMR (500 MHz, CDCl₃) chemical shifts and coupling constants for theprotected pseudo-pentasaccharide 16c.^(a) Ring H1 H2 H3 H4 H5 H5′ H6 H6′A 6.04 3.38-3.42 5.51 5.08 4.50-4.59 3.42-3.53 3.42-3.53 d m dd t m m mJ = 4.0 J = 9.5, 10.5 J = 9.5 C 5.23 4.50-4.59 4.13-4.16 4.28 3.734.13-4.16 d m m ddd dd m J = 3.0 J = 3.0, J = 4.5, 5.0, 6.5 12.5 D 4.683.25 5.08 4.70 4.03 3.42-3.53 3.38-3.42 d bs t bs ddd m m J = 2.5 J =3.0 J = 1.5, 5.0, 6.0 E 5.62 4.64 6.62 5.62 4.13-4.16 4.40 4.50-4.59 ddd dd dd m dd m J = 8.5 J = 8.5, J = 9.0, 10.5 J = 9.5, J = 3.0, 11.013.0 12.5 H1 H2eq H2ax H3 H4 H5 H6 B 3.38-3.42 2.12 1.75 3.42-3.53 4.923.89 3.82 m dt ddd m t t t J = 4.5, J₁= J₂ = J₃ J = 10.0 J = 8.5 J = 8.513.0 = 12.5^(a)Values of chemical shifts are in ppm and values of couplingconstants are in Hz. The additional peaks in the spectrum wereidentified as follow: δ 1.98 (s, 3H, acetate), 2.09 (s, 3H, acetate),2.10 (s, 3H, acetate), 2.16 (s, 3H, acetate), 2.165 (s, 3H, acetate),2.19 (s, 3H, acetate) 4.17 (s, 2H, chloroacetate), 7.24-7.92(m, 14H,aromatic).

TABLE 7

¹H NMR (500 MHz, CDCl₃) chemical shifts and coupling constants for theprotected pseudo-pentasaccharide 16d.^(a) Ring H1 H2 H3 H4 H5 H5′ H6 H6′A 6.06 3.21-3.31 3.71 4.89-4.95 3.40 3.27-3.34 3.27-3.34 d m dd m ddd mm J = 4.0 J = 9.5, J = 3.5, 5.0, 11.0 10.0 C 5.33 4.79 4.35-4.374.28-4.31 3.45 3.74 d dd m m dd dd J = 4.5 J₁ = J₂ = J = 3.5, 10.0 J =2.0, 4.5 10.0 D 4.83 3.27-3.34 4.66 4.97 4.05 3.59 3.74 d m bs dd ddd dddd J = 4.0 J₁ = J₂ = 4.5 J = 2.0, 5.0, J = 8.5, J = 4.0, 6.5 13.0 12.0 E5.95 4.92 4.92 4.12-4.20 4.12-4.20 4.35-4.37 d dd dd m m m J = 4.5 J =3.5, 5.5 J = 3.5, 5.5 H1 H2eq H2ax H3 H4 H5 H6 B 3.40 2.33 1.58 3.504.93 3.88 3.66 ddd dt ddd ddd t t t J = 4.5, J = 4.0, J₁ = J₂ = J₃ J =4.0, J = 12.0 J = 9.5 J = 9.0 10.5, 12.0 13.5 = 12.5 10.0, 12.5^(a)Values of chemical shifts are in ppm and values of couplingconstants are in Hz. The additional peaks in the spectrum wereidentified as follow: δ 2.00 (s, 3H, acetate), 2.05 (s, 6H, 2 acetates),2.06 (s, 3H, acetate), 2.07 (s, 3H, acetate), 2.10 (s, 3H, acetate),2.11 (s, 3H, acetate), 2.13 (s, 3H, acetate), 2.16 (s, 3H, acetate).

TABLE 8

¹H NMR (500 MHz, CDCl₃) chemical shifts and coupling constants for thetitled structure.^(a) Ring H1 H2 H3 H4 H5 H5′ H6 H6′ A 6.15 3.44-3.625.52 5.18 4.60-4.64 3.44-3.62 3.44-3.62 d m t t m m m J = 3.5 J = 10.5 J= 10.0 C 5.24 4.47 4.16 4.33 3.73 4.22 d dd t bdd dd dd J = 2.5 J = 2.5,J = 4.5 J = 3.5, J = 2.5, 4.5 11.0 11.0 D 4.99 3.23 3.86 4.66-4.703.98-4.03 3.41-3.52 3.44-3.62 d bs bs m m m m J = 1.0 E 4.60-4.644.66-4.70 6.26 5.57 4.10 3.27 3.44-3.62 m m t t ddd dd m J = 10.5 J =9.5 J = 2.5, J = 5.5, 7.5, 13.5 13.0 H1 H2eq H2ax H3 H4 H5 H6 B 3.372.41 1.81 3.44-3.62 3.98-4.03 3.82 4.96 ddd dt ddd m m t t J = 4.5, J =4.5, J₁ = J₂ = J₃ J = 9.0 J = 10.0 10.5, 13.5 13.0 = 12.5^(a)Values of chemical shifts are in ppm and values of couplingconstants are in Hz. The additional peaks in the spectrum wereidentified as follow: δ 1.94 (s, 3H, acetate), 2.08 (s, 3H, acetate),2.10 (s, 3H, acetate), 2.14 (s, 3H, acetate), 2.17 (s, 3H, acetate),2.18 (s, 3H, acetate), 7.29-8.10 (14H, aromatic protons).

TABLE 9

¹H NMR (500 MHz, CDCl₃) chemical shifts and coupling constants for thetitled structure.^(a) Ring H1 H2 H3 H4 H5 H5′ H6 H6′ A 6.14 328-3.355.47 5.09 3.98 3.41-3.52 3.41-3.52 d m t t ddd m m J = 3.5 J = 9.0 J =9.5 J = 3.0, 3.0, 10.0 C 5.17 4.71 4.27 4.08 4.25 3.55-3.65 s d dd m ddm J = 5.0 J = 4.5, J = 1.0, 7.0 11.0 D 4.36 4.92 4.60 3.41-3.52 3.133.28-3.35 3.41-3.52 d t bs m bt m m J = 1.5 J = 2.0 E 4.66 5.45 5.593.99 3.55-3.65 4.45 4.40 d t t t m dd dd J = 8.0 J = 10.0 J = 10.0 J =10.0 J = 1.5, J = 5.0, 11.5 12.0 H1 H2eq H2ax H3 H4 H5 H6 B 3.28-3.352.34 1.65 3.41-3.52 3.93 377 3.86 m dt ddd m t t t J = 4.0, J₁ = J₂ = J₃J = 9.5 J = 9.0 J = 9.5 13.0 = 12.5^(a)Values of chemical shifts are in ppm and values of couplingconstants are in Hz. The additional peaks in the spectrum wereidentified as follow: δ 1.98 (s, 3H, acetate), 2.06 (s, 3H, acetate),2.07 (s, 3H, acetate), 2.10 (s, 3H, acetate), 2.12 (s, 3H, acetate),2.14 (s, 3H, acetate), 2.19 (s, 3H, acetate), 7.60 (t, J = 7.5 Hz, 2H,meta benzoyl protons), 7.69 (t, J = 8.0 Hz, 2H, meta benzoyl protons),7.77 (t, J = 7.5 Hz, 1H, para benzoyl proton), 7.84 (t,# J = 7.5 Hz, 1H, para benzoyl proton), 8.15 (d, J = 8.5 Hz, 1H, orthobenzoyl proton). 8.30 (d, J = 7.5 Hz, 1H, ortho benzoyl proton).

TABLE 10

¹H NMR (500 MHz, CDCl₃) chemical shifts and coupling constants for thetitled structure.^(a) Ring H1 H2 H3 H4 H5 H5′ H6 H6′ A 5.73 3.09-3.133.77 3.26-3.3 3.98 3.47 3.39 d m t m ddd bd dd J = 4.0 J = 8.5 J = 2.0,J = 5.5, 5.5, 8.5 14.5 C 5.36 4.77 4.36 4.23 3.71 3.86 d t t dd dd m J =4.5 J = 4.5 J = 4.7 J = 4.0, J = 4.5, 6.0 11.5 D 4.99 3.66 3.86 3.273.41-3.52 3.41-3.52 3.41-3.52 d bs bs bs m m m J = 1.0 E 5.86 4.85 4.853.33-3.39 3.42-3.46 3.33-3.39 3.23 d dd dd m m m dd J = 5.5 J₁ = J₂ = J= 5.2, 5.3 J = 3.0, 4.5 17.0 H1 H2eq H2ax H3 H4 H5 H6 B 3.26-3.36 2.121.31 3.26-3.36 3.51-3.61 3.51-3.61 3.41 m dt ddd m m m t J = 4.0, J₁ =J₂ = J₃ J = 9.0 13.0 = 12.5^(a)Values of chemical shifts are in ppm and values of couplingconstants are in Hz. The additional peaks in the spectrum wereidentified as follow: δ 1.99 (s, 3H, acetate), 2.02 (s, 3H, acetate),2.03 (s, 3H, acetate), 2.04 (s, 3H, acetate), 2.12 (s, 3H, acetate),2.13 (s, 3H, acetate), 7.29-8.10 (10H, meta aromatic benzoyl protons).

TABLE 11 16h

¹H NMR (500 MHz, CDCl₃) chemical shifts and coupling constants for thetitled structure.^(a) Ring H1 H2 H3 H4 H5 H5′ H6 H6′ A 5.91 3.24-3.355.49 4.93-5.04 4.43-4.46 3.24-3.35 3.24-3.35 d m dd m m m m J = 3.5 J₁ =J₂ = 10.5 C 5.34 4.93-5.04 4.07-4.24 4.07-4.24 3.59-3.66 4.07-4.24 d m mm m m J = 1.0 D 4.89 3.24-3.35 4.93-5.04 4.71-4.72 4.07-4.24 3.48 3.19 dm m m m dd dd J = 1.5 J = 5.0, J₁ = 4.0 13.0 J₂ = 13.0 E 5.17 5.23 4.614.29-4.32 4.43-4.46 4.07-4.24 s d dd m m m J = 4.5 J = 4.5, 7.5 F4.93-5.04 3.24-3.35 4.93-5.04 4.71-4.72 4.07-4.24 3.59-3.66 3.24-3.35 mm m m m m m H1 H2eq H2ax H3 H4 H5 H6 B 3.59 2.39 1.64 3.48 3.71 3.914.93-5.04 m dt ddd m t t m J = 4.5, J = J₂ = J₃ ₌ J = 9.0 J = 9.0 13.013.0^(a)Values of chemical shifts are in ppm and values of couplingconstants are in Hz. The additional peaks in the spectrum wereidentified as follow: δ 2.07 (s, 3H, acetate), 2.09 (s, 3H, acetate),2.11 (s, 3H, acetates), 2.12 (s, 3H, acetate), 2.14 (s, 3H, acetate),2.16 (s, 6H, acetate), 2.17 (s, 6H, acetates), 2.18 (s, 6H, acetate),2.19 (s, 6H, acetate).

TABLE 12

¹H NMR (500 MHz, CDCl₃) chemical shifts and coupling constants for thetitled structure.^(a) Ring H1 H2 H3 H4 H5 H5′ H6 H6′ A 5.93 3.26-3.455.48 5.02-5.05 4.44 3.26-3.45 3.26-3.45 d m t m ddd m m J = 3.5 J = 10.0J = 3.5, 6.5, 10.0 C 5.28 4.69-4.71 5.48 4.30 4.05-4.08 4.05-4.08 d m tdd m m J = 4.0 J = 6.0 J = 4.0, 8.0 D 4.81 3.26-3.45 4.69-4.71 5.02-5.053.64 3.26-3.45 3.26-3.45 s m m m dd m m J = 7.5, 13.0 H1 H2ax H2eq H3 H4H5 H6 B 3.26-3.45 1.59 2.39 3.49 3.70 3.88 3.69 m ddd m ddd t t t J₁ =J₂ = J₃ = J = 4.5, J = 9.5 J = 9.0 J = 10.5 12.5 8.5, 16.5^(a)Values of chemical shifts are in ppm and values of couplingconstants are in Hz. The additional peaks in the spectrum wereidentified as follow: δ 2.05 (s, 3H, acetate), 2.08 (s, 6H, acetate),2.14 (s, 3H, acetate), 2.15 (s, 3H, acetate), 2.16 (s, 3H, acetate),2.36 (s, 3H, MeSAc).

TABLE 13 2

¹H NMR (500 MHz, CDCl₃) chemical shifts and coupling constants for thetitled structure.^(a) Ring H1 H2 H3 H4 H5 H5′ H6 H6′ A 5.94 3.20-3.285.45 4.96-4.98 4.40 3.20-3.28 3.31 d m t m ddd m dd J = 4.0 J = 10.0 J =3.0, 6.0, J = 2.0, 10.0 13.0 C 5.28 4.83 4.29 4.11 2.76 2.91 d s t m dddddd J = 2.5 (broad) J = 5.5 J = 7.5, J = 3.0, 14.0, 18.5 8.5, 18.0 D4.99 3.47 4.96-4.98 4.65 4.06 3.20-3.28 3.54 d dd m s m m dd J = 1.5 J =2.5, J = 8.0, 13.5 13.0 H1 H2eq H2ax H3 H4 H5 H6 B 3.40 2.51 1.56 3.503.67 3.87 4.91 ddd dt ddd ddd t t t J = 4.5, J = 4.0, J₁ = J₂ = J₃ = J =4.5, J = 9.0 J = 9.0 J = 9.5 10.5, 14.5 13.0 12.5 10.0, 14.0^(a)Values of chemical shifts are in ppm and values of couplingconstants are in Hz. The additional peaks in the spectrum wereidentified as follow: δ 1.78 (t, J = 8.0 Hz, 1H, SH), 2.00 (s, 3H,acetate), 2.04 (s, 3H, acetate), 2.05 (s, 3H, acetate), 2.10 (s, 6H,acetate), 2.12 (s, 3H, acetate),

TABLE 14 16i

¹H NMR (500 MHz, CDCl₃) chemical shifts and coupling constants for thetitled structure.^(a) Ring H1 H2 H3 H4 H5 H5′ H6 H6′ A 5.91 3.24-3.335.44 4.96-5.04 3.41 3.24-3.33 3.24-3.33 d m t m ddd m m J = 5.5 J = 10.5J = 3.0, 5.5, 9.5 C 5.23 4.87-4.91 4.28 4.28 3.14 3.01 d m bs bs m dd J= 1.0 J = 7.5, 13.5 D 4.87-4.91 3.24-3.33 4.96 4.64 3.63-3.67 3.143.52-3.56 m m s s m m m E 5.61 5.76 5.95 4.65-4.73 4.65-4.73 4.60 d dddd m m dd J = 2.0 J = 2.0, J = 5.5, 6.0 J = 5.0, 5.0 12.0 H1 H2eq H2axH3 H4 H5 H6 B 3.24-3.33 2.51 1.55 3.41 3.76 3.62 4.87-4.91 m dt ddd dddt t m J = 5.0, J₁ = J₂ = J₃ = J = 4.5, J = 9.0 J = 9.0 13.0 12.5 10.0,14.0^(a)Values of chemical shifts are in ppm and values of couplingconstants are in Hz. The additional peaks in the spectrum wereidentified as follow: δ 1.99 (s, 3H, acetate), 2.02 (s, 3H, acetate),2.03 (s, 3H, acetate), 2.04 (s, 3H, acetate), 2.12 (s, 3H, acetate),2.13 (s, 3H, acetate), 7.29-8.10 (10H, aromatic benzoyl protons).

TABLE 15 XI

¹H NMR (500 MHz, D₂O pH = 3.04) chemical shifts and coupling constantsfor the titled structure.^(a) Ring H1 H2 H3 H4 H5 H5′ H6 H6′ A 6.033.43-3.47 3.95-3.99 3.23-3.39 3.80-3.89 3.14 3.23-3.39 d m m m m dd m J= 4.0 J = 7.5, 13.5 C 5.31 4.37 4.40 4.26 3.53 3.61-3.65 bs bs bt bdd ddm J = 5.0 J = 6.0, 12.5 D 5.19 3.43-3.47 4.11-4.15 3.68 4.21 3.23-3.393.23-3.39 bs m m bs bt m m E 4.96 3.95-3.99 4.03 3.80-3.89 3.36-3.392.86 d m t m m dd J = 6.0 J = 4.0 J = 8.0, 13.5 H1 H2eq H2ax H3 H4 H5 H6B 3.23-3.39 2.35 1.95 3.43-3.47 3.61-3.65 3.80-3.89 4.11-4.15 m bdt dddm m m m J₁ = J₂ = J₃ = 12.5^(a)Values of chemical shifts are in ppm and values of couplingconstants are in Hz.

TABLE 16

¹H NMR (500 MHz, CDCl₃) chemical shifts and coupling constants for thetitled structure.^(a) Ring H1 H2 H3 H4 H5 H5′ H6 H6′ A 6.21 3.31-3.385.47 5.09 3.98 3.40-3.48 3.42 d m t t ddd m dd J = 3.5 J = 9.0 J = 9.5 J= 3.0, J = 2.5, 3.0, 10.0 11.5 C 5.22 4.74 4.27 4.30 4.25 3.58-3.67 d ddd dd dd m J = 1.0 J = 5.0 J = 5.0, 6.5 J = 5.0, J = 1.0, 7.5 11.0 D4.38 4.89 4.60 3.41-3.52 3.15 3.31-3.38 3.41-3.52 d t bs t m m J = 1.5 J= 2.0 m J = 2.5 E 4.72 5.06 5.34 5.18 3.58-3.67 4.47 4.42 d t t t m dddd J = 9.5 J = 10.0 J = 10.0 J = 10.0 J = 1.5, J = 4.0, 11.5 11.5 H1H2eq H2ax H3 H4 H5 H6 B 3.31-3.38 2.37 1.58 3.41-3.52 3.93 3.77 3.86 mdt ddd m t t t J = 4.5, J₁ = J₂ = J₃ = J = 9.5 J = 9.0 J =+09 .5 12.512.5^(a)Values of chemical shifts are in ppm and values of couplingconstants are in Hz. The additional peaks in the spectrum wereidentified as follow: δ 1.97 (s, 3H, acetate), 1.99 (s, 3H, acetate)2.08 (s, 3H, acetate), 2.10 (s, 6H, acetate), 2.13 (s, 3H, acetate),2.14 (s, 3H, acetate), 2.16 (s, 6H, acetate), 2.18 (s, 3H, acetate),

TABLE 17

¹H NMR (500 MHz, CDCl₃) chemical shifts and coupling constants for thetitled structure.^(a) Ring H1 H2 H3 H4 H5 H5′ H6 H6′ A 5.95 3.49-3.683.91 3.15-3.44 3.71-3.91 3.49-3.68 3.15-3.44 d m t m m m m J = 3.5 J =10.0 C 5.28 4.37 4.64 4.08-4.25 3.49-3.65 4.08-4.25 d d m m m m J = 2.5J = 2.0 D 5.15 3.15-3.44 4.08-4.25 3.49-3.68 4.08-4.25 3.15-3.443.15-3.44 d m m m m m m J = 1.0 E 4.36 3.15-3.44 3.15-3.44 3.15-3.443.49-3.68 3.71-3.91 3.44-3.62 d m m m m m m J = 8.0 H1 H2eq H2ax H3 H4H5 H6 B 3.15-3.44 2.32 1.94 3.15-3.44 3.15-3.44 3.71-3.91 4.08-4.25 m mddd m m m m J₁ = J₂ = J₃ = 12.5^(a)Values of chemical shifts are in ppm and values of couplingconstants are in Hz.

EXAMPLE 4 Additional Synthetic Strategies for Other Selected Compoundsof the Present Invention

Example 3 above related to a synthetic strategy for specific selectedcompounds according to the present invention. However, this strategy mayoptionally be generalized to obtain any member of the set4-set5structures shown in FIG. 9.

FIG. 13 shows the overall synthetic protocol for the assembly ofset4-set5 structures. The protected perazido-neomycin B (compound 17 a,FIG. 13) can be sufficiently hydrolyzed in the presence of TolSH andBF₃-OEt₂ to yield the neamine fragment 18 and the thioglycoside 19 a in90% and 82% yields, respectively (38). Inversion of configuration at C5of 18 provides the protected epi-neamine 20. While this step can beaccomplished in various methods, the approach of Moriarty et al (39),that uses triflation of the alcohol followed by treatment with sodiumnitrite, has been proved to be very successful when examined fordifferent oligosaccharides. The 5-epi-neamine derivative 20 can beeasily transformed to the corresponding 5-thio-neamine 21 in two simplesteps: Conversion of the 5″ hydroxyl in to the corresponding S-acetyl bythe Mitsunobu procedure, followed by S-deacetylation using hydraziniumacetate in DMF. Treatment of 20 with the thioglycoside 19 a will providethe core structure of the protected 5-epi-neomycin B (22 a), which aftersubsequent deprotection steps will afford the epi-neomycin B. Similarly,treatment of the protected 5-thio-neamine 21 with the correspondingbromide of 19 a and deprotection steps of the intermediate 23 a willafford the 5-thio-neomycin B.

In summary, the synthetic strategy outlined in FIG. 13 involves theconversion of the natural neomycin B to the corresponding 5-epi-neomycinB (22 a) and 5-thio-neomycin B (23 a) with a maximum efficiency: no lossof neomycin fragments and no addition of extra sugars. This strategy isvery advantageous, especially because of the relatively low cost of thecommercial neomycin B. In addition, this strategy also provides anefficient method for the preparation of epi-ribostamycin and5-thio-ribostamycin (FIG. 1). Thus, coupling of 20 with 8 a, followed bysimple deprotection steps as outlined above will result the5-epi-ribostamycin. Similarly, coupling of 21 with the correspondinganomeric bromide of 8 a after subsequent deprotection will afford a5-thio-ribostamycin.

Furthermore, by starting this pathway with compound 17 b (instead of 17a) it is possible to generate the corresponding 5-epi and 5-thioderivatives of neomycin, 22 b and 23 b, respectively. Selectivedeprotection of the silyl group in these compounds will result thecorresponding C5″—OH derivatives, 22 (R═H) and 23 (R═H), which will beused as common acceptors for the preparation of set4 and set5 compounds.

Optionally, it is possible to further modify these structures and tothereby generate a library of set4 and set5 compounds. For this purposeoptionally and preferably the general strategy outlined in FIG. 8 forthe preparation of set1-set3 compounds is followed, but instead of 1-3as acceptors compounds 22 and 23 are employed.

EXAMPLE 5 Antibiotic Activities of the Compounds According to thePresent Invention

The new analogs have been tested for antibacterial activities againstboth Gram-negative and Gram-positive bacteria including pathogenic andresistant strains by determining minimal inhibitory concentrations(MICs). FIG. 16 shows the structures of neomycin B (Compound I) andCompounds II-XIII according to the present invention, which were testedas described in greater detail below.

In addition to the standard resistant strains, for which theirmechanisms of resistance are well known, the activity of these neomycinB derivatives have been studied on multiple antibiotic resistant“natural” strains collected from human and farm origin. One exemplarymodel is the food-borne pathogen Salmonella, since it is among theleading cause of foodborne disease, foodborne-related hospitalizationand foodborne-related deaths. The salmonellae bacteria are responsiblefor an estimated 16 million annual instances of typhoid fever, primarilyin developing countries, and untold millions of cases of gastroenteritisin both industrialized and developing countries. This is a zoonoticpathogen that usually exposed to a variety of antibiotics in the farm,including a wide range of aminoglycosides.

This Example describes experiments which were performed to test theefficacy of the compounds of the present invention against differentmicroorganisms, including strains of those microorganisms which werealready shown to be antibiotic resistant.

The compounds were tested for antibacterial activities against bothGram-negative and Gram-positive bacteria, including pathogenic andresistant strains, and the minimal inhibitory concentrations (MIC) weredetermined using a microdilution assay with neomycin B and kanamycin ascontrols (see Phillips, I.; Williams, D. In Laboratory Methods inAntimicrobial Chemotherapy; Gerrod, L., Ed.; Churchill LivingstonePress: Edinburg 1978; pp 3-30).

Resistant strains included E. coli XL1(pET9d), Pseudomonas aeruginosa(ATCC 27853), and Salmonella virchow (SV49). E. coli XL1-(pET9d) is anantibiotic-sensitive laboratory strain of E. coli that harbors plasmidpET9d with the cloned orf2 gene, which codes for aminoglycoside kinaseAPH(3′). P. aeruginosa is a Gram-negative pathogen. The aph(3′)-IIbgene, which codes for APH(3′), is a chromosomal gene that was found inmany clinical isolates of P. aeruginosa, including the ATCC 27853strain, and likely accounts at least partly for the resistance ofPseudomonas to aminoglycosides (Hachler, H.; Santanam, P.; Kayser, F. H.Antimicrob. Agen. Chemother. 1996, 40, 1254-1256). S. virchow (SV49) isa clinical multidrug-resistant strain obtained from poultry and found tobe resistant to streptomycin, tetracycline, ampicillin, sulfa,kanamycin, and neomycin. The mechanism(s) of resistance of this strainis still not known.

The results are shown in Table 18 below.

From the MIC values, it turns out that among the four analogs, onlyCompound V having a ribose substituent at ring E is as potent asneomycin B against E. coli strains. The activity of this analogueagainst E. coli XL1(pET9d) having kanamycin resistance is even moreimpressive, exhibiting better activity than neomycin B. The analogueCompound V is also effective against Gram-positive bacteria,Staphylococcus epidermidis and Bacillus subtilis. Furthermore, CompoundV demonstrates better activity than other analogues against pathogenicbacterium Salmonella virchow that is resistant to kanamycin and neomycinB. In this case Compound V is about 5 times more effective thankanamycin and 2 times more effective than neomycin B. The susceptibilityof enterobacterium Pseudomonas aeruginosa was also examined, which isoften very difficult to treat, sometimes requiring use of a combinationof aminoglycosides with other antibiotics (Haddad, J.; Kotra, L. P.;Liano-Sotel, B.; Kim, C.; Azucena, E. F., Jr.; Liu, M.; Vakulenko, S.B.; Chow, C. S.; Mobashery, S. J. Am. Chem. Soc. 2002, 124, 3229-3237).Interestingly, in this particular case, while Compound V demonstratesactivity close to that of neomycin B, while the 2-glucosamino derivativeCompound IV is even more effective than Compound V and the diaminoderivative Compound III is superior to both. TABLE 18 MICs of CompoundI-V against Various Bacterial Strains MIC (μg/mL) bacterial strainKAN^(a) I II III IV V E. coli (R47-100) ND^(b) 4-5.5 85 40-50 35-404.5-6 E. coli (ATCC 25922) ND 8-10 95 40-50 25-30 10-11 E. coli XL1 blue(pET9d) 260- 50-60 >200 >200 >200 35-45 270 Staphylococcus epidermidis(ATCC ND 0.3-0.4 5.5-7 1.5- 1.4- 0.2- 12228) 1.8 1.8 0.4 Bacillussubtilis (ATCC 6633) ND 0.8-0.9 8.5-10 3.5-4 1.4- 0.6- 1.8 0.8Salmonella virchow (SV49) 500- 200- >1250 >1250 >1250 75- 570 250 125Pseudomonas aeruginosa (ATCC 450- 55-60 110- 30-35 40-50 60-65 27853)500 130^(a)KAN = kanamycin. ^(b)ND = not determined.

The observed preliminary data obtained with Compounds II-V indicatethat, without wishing to be limited by a single hypothesis, merelyincreasing the number of amino groups on the natural drug may not leadto an increase in antibacterial activity, even though the bindingaffinity of these analogues to RNA is likely to increase in vitro.However, the excellent activities observed for the amino derivatives IIIand IV against Pseudomonas but significantly weak activities againstother bacterial strains imply that the structural and functionalrequirements for this family of drugs are not similar in order to reachanalogous high antibacterial performance against different organisms,again without wishing to be limited by a single hypothesis.

Additional data has been obtained with other compounds according to thepresent invention, as shown with regard to Table 19 below. Theseexperiments were performed in a manner similar to those described above.TABLE 19 MICs of Compounds VI-XI against various bacterial strains MIC(μg/mL) Bacterial strain KAN^(a) I VI VII VIII IX X XI E. coli (R47-100)ND^(b) 4-5.5 35-50 45-50 50-70 90-100 150-200 30-40 E. coli (ATCC 25922)ND 8-10 35-40 45-60 30-50 100-150 100-150 25-40 E. coli XL1 blue(pET9d)^(c) 260-270 50-60 200-250 250-400 150-200 700-800 250-500 >200Staphylococcus ND 0.3-0.4 4-5 4.5-5.5 5.5-6.0 10-11 4-5 1.5-3epidermidis (ATCC 12228) Bacillus subtilis ND 0.8-0.9 5-7 6-7 5-7 10-115-10 1-2.5 (ATCC 6633) Salmonella virchow 500-570200-250 >1000 >1000 >1000 >800 >1000 >250 (SV49) Pseudomonas 400-50055-60 55-60 45-50 10-15 350-400 40-60 200-400 aeruginosa (ATCC 27853)^(a)KAN, kanamycin; ^(b)ND, not determined.

As can be seen from these additional results, several of the compoundsprovide results which are at least as good as kanamycin and/or neomycinB for particular strains, such as certain strains of E. coli. CompoundsVI-VIII and X provided improved results over kanamycin and similarresults to neomycin B for other strains, such as resistant forms ofPseudomonas aeruginosa. Indeed for this strain, Compound VIII providedsignificantly better results than either kanamycin or neomycin B.Overall good results against different strains were demonstrated for allof the compounds for at least certain strains.

Overall, the neomycin B derivatives prepared in this study represent anew class of branched aminoglycoside antibiotics that show antibacterialactivity superior to that of neomycin B and/or kanamycin againstpathogenic and resistant bacterial strains, although the breadth ofactivity across different strains differed between the compounds.

EXAMPLE 6 Treatment of Genetic Disorder With Compounds According to thePresent Invention

The previous Example discussed the antibiotic activities of someexemplary compounds of the present invention. However, these compoundsare expected to have other effects as well, some of which are discussedbelow.

One illustrative use of the compounds of the present invention is fortreatment of genetic disorder, such as cystic fibrosis for example. Thetreatment of cystic fibrosis with the aminoglycosaccharide gentamicinhas been shown (Wilschanski et al, “Gentamicin-induced correction ofCFTR function in patients with cystic fibrosis and CFTR stop mutations”,New Eng. J. Med., vol 349, pp. 1433-41 Oct. 9, 2003; hereby incorporatedby reference as if fully set forth herein). It is believed that thiseffect is obtained by blocking a premature stop codon which leads to ashortened version of CFTR (cystic fibrosis transmembrane conductanceregulator); this mutation causes the effects of cystic fibrosis, whichcause the lungs of the affected subject to fill with mucous, leading tobacterial infection, severely reduced pulmonary function and oftenpremature death. Blocking the premature stop codon causes “readthrough”, such that a longer protein is transcribed which has at leastadditional activity compared to the mutated protein. In the previouslydescribed reference, treatment with gentamicin was shown to result infull length CFTR in a number of patients.

Without wishing to be limited by a single hypothesis, it is believedthat as the compounds of the present invention are also aminoglycosidederivatives, the compounds of the present invention should also beuseful for treatment of cystic fibrosis. Such treatment may be effectivefor a number of reasons, including but not limited to, one or more ofreduction or elimination of bacterial infection through the antibioticeffect of the compounds according to the present invention; and/or alsoblocking the premature stop codon.

Treatment would preferably include administration of a therapeuticallyeffective amount of a compound according to the present invention to asubject. Dosing and administration routes could easily be determined byone of ordinary skill in the art, and would optionally include suchroutes as oral, topical, nasal, inhaled, optical, parenteral and soforth, as described in greater detail below; however, for cysticfibrosis treatment, optionally and preferably treatment would includeadministration of the compound according to the present inventiondirectly to the lungs, for example through an inhaled spray or mist,and/or powder inhaler. Preferably, the compound would be provided in asuitable formulation, also as described in greater detail below. Thecompound may also optionally be combined with other type(s) of treatmentfor cystic fibrosis, for example by including treatment with one or moreother medications that are known in the art.

Non-limiting examples of other genetic disorders for which treatmentwith a compound according to the present invention may be useful includeDuchenne's muscular dystrophy or Hurler's syndrome which are alsocharacterized by truncation mutations.

EXAMPLE 7 Other Activities of the Compounds According to the PresentInvention

The previous Example discussed the antibiotic activities and alsoanti-cystic fibrosis activity of some exemplary compounds of the presentinvention. However, these compounds are expected to have other effectsas well, some which are discussed below.

Aminoglycoside variants as potential ribonucleases

Since aminoglycoside antibiotics exert their antibacterial activity byselectively recognizing and binding to a ribosomal RNA, it ishypothesized that the combination of this already existing recognitionelement in natural drugs with a catalytic element into a single moleculewould significantly increase the activity of the resulted structure. Thefollowing observations supported this hypothesis. First, Wong andco-workers demonstrated a nearly linear relationship between the IC₅₀ ofin vitro translation inhibition and the MIC values for a series ofnatural aminoglycosides and their synthetic analogs (12 e, 14). Ingeneral, MIC values were at about 100-fold higher concentrations thanthe corresponding IC₅₀ values.

To explain these differences it was suggested that since the ribosomalRNA is the most dominant RNA in the cell, at low drug concentrations, atight ribosome-binding drug titrates only a small fraction of the verylarge number of ribosomes and only at higher concentrations of drug areall ribosomes saturated and protein synthesis impaired. These data implythat an increasing binding affinity of the drug to target RNA should notmerely result in better antibiotic function in the sense of requiredadministered dose of the drug, and that most potent ribosome-targetingantibiotics could be envisioned if they were designed to be catalyticinhibitors (12 e, 14).

Second, several examples of site-directed RNA cleaving agents thatcombine a reactive moiety capable of cleaving phosphodiester bond with arecognition element capable of sequence-specifically hybridizing totarget RNA, have been reported (41). Third, in analogy to earlierobservations in which several simple oligoamines, (42), as well as basicpolypeptides (43) have been shown to catalyze RNA hydrolysis, it waslikely that aminoglycosides that represent polycationic molecules couldexhibit similar effect. Indeed, it has recently been shown that neomycinB, which has three times as many amines as 1,3-propanediame, catalyzeshydrolysis of adenylyl(3′-5′)-adenosine (ApA) 3-fold faster than1,3-propanediamine (44). Neomycin B consists of themeso-1,3-diaminocyclitol (2-deoxystreptamine) ring for which the pK_(a)values of 5.74 and 8.04 were reported. This may lead to a higherpopulation of a monocationic form at a given pH compared to1,3-propanediamine, and therefore to a faster hydrolysis.

However, the observed first-order rate constants for neomycin was over1000-fold lower than those reported for natural ribozymes. It is clearthat further increase of the catalytic activity of naturalaminoglycosides should be plausible if the molecular design will be moreprecise. The simplicity and stability of aminoglycosides, in conjunctionwith the recent progress in 3D structure determination ofaminoglycosides bound to rRNA, are undoubtedly advantageous for thispurpose.

Design and synthesis of neomycin variants as potential ribonucleases

Based on the results obtained with various oligoamines as a motif forthe molecular recognition and hydrolysis of the phosphodiester bond ofRNA, briefly introduced in the previous section, two series of neomycinanalogs are prepared: (1) structures of set1-set5 that contain1,2-diamino and 1,3-diamino moieties as “catalytic warheads”; (2)structures of set5 and set6 that represent pseudo-hexasaccharidevariants.

(1) Variants with “catalytic warheads.” For this purpose the diaminebuilding blocks 5 f, 6 f, 7 a, (FIG. 7) and 24 (FIG. 14) were speciallydesigned. Structures 7 a and 24 consist of 1,2-diamino moieties in a cisconfiguration. Such vic-cis-diamino moieties in 7 a and 24 exhibit rigidspatial orientation of two neighboring amino groups and might be moreadvantageous for catalysis than highly flexible amines in1,2-ethylenediamine. In addition, N-C-C-N torsion angle inribofuranoside 24 (eclipsed relationship) is different than that inallopyranoside 7 a (gauche relationship). Structures 5 f and 6 f contain1,3-diamino moieties and have gluco and galacto configurations,respectively. It is noteworthy that although 1,3-diamino moiety is verycommon in aminoglycoside antibiotics, this moiety is mostly present inthe 2-deoxystreptamine unit (meso-1,3-diamino cyclitol, ring B inneomycin B) and is different from the flexible diamine such as in 5 fand 6 f. Therefore, investigation of 5 f and 6 f, along with 7 a and 24,is very challenging from both points of view: they represent new“catalytic warheads” for the cleavage of phosphodiester bond, and theirincorporation into appropriate oligosaccharides, may result novelantibiotics.

A description of this synthetic scheme is described with regard to FIG.14.

These monosaccharide building blocks can be incorporated into the abovedesigned set1-set5 structures as variable sugar rings to yieldcorresponding pseudo-pentasaccharides, which will be tested forribonuclease activity as outlined below.

(2) pseudo-hexasaccharide variants. Electrostatic interactions have beenshown to be critically important in RNA binding (45). Increasing thenumber of positively charged ammonium groups in ligands resultedenhanced binding affinities by RNA host. Since binding affinity ofsubstrate to the catalyst is very important and strongly contributes tothe overall efficiency of any catalytic system, it is clear that byincreasing the binding affinities of our designed structures to RNAsubstrate we should subsequently increase their probability ascatalysts. Therefore, in attempts to improve the catalytic power of theabove designed pseudo-pentasaccharides (set1-set5), a new set, set6, ofthe pseudo-hexasaccharides are prepared (FIGS. 14 and 15). The twopseudo-hexasaccharides 25 and 26 can be easily assembled by coupling ofthe disaccharide donor 19 a (FIG. 13) with either 1 or 2 as acceptorsand subsequent deprotection steps as illustrated in FIG. 14. (Noteadded: As an illustrative example, compound 25 (which is also the finalproduct Compound X in FIG. 16) has been successfully synthesized and thedata of this synthesis and antibacterial tests are summarized in theprevious sections.) These two compounds remain the original neomycinstructure, so that the likelihood of their binding to the rRNA A-site isvery high. The other structure of set 6, compound 27, has theepi-neomycin core, and can be easily assembled from the neomycinfragments discussed above. Thus, coupling of neamine derivative 20 with19 b will afford the corresponding epi-neamine derivative, which afterdeprotection of the primary alcohol, coupling with 19 a, anddeprotection steps will furnish the epi-neomycin hexasaccharidederivative 27 (FIG. 15).

EXAMPLE 8 Antibacterial Activity Against Bacillus anthracis andInhibition of Anthrax Lethal Factor By the Compounds of the PresentInvention

Example 5 above showed that the compounds according to the presentinvention can be used for treatment of a subject suffering frominfection by an infectious microorganism, including gram-negative andgram-positive bacteria for example.

This Example provides an illustrative, non-limiting example of use ofselected compounds according to the present invention against theGram-positive bacterium, Bacillus anthracis for treatment of anthrax,and for inhibition of Anthrax Lethal Factor.

The new derivatives of neomycin B, Compounds II-XIII of FIG. 16, weresynthesized according to the general strategy described in Example 1 forcompounds of Formula I (FIG. 8), via the corresponding intermediates.The synthetic strategy and preparation of the exemplary intermediatesare set forth in Example 1.

Using an in vitro fluorescent assay (22), the novel Compounds II-XIIIaccording to the present invention were examined for the proteaseactivity inhibition of LF, at both high and low salt concentrations.

Low-salt conditions comprised potassium2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) bufferat pH 7.4 (10 mM), LF (about 33 nM), varying concentrations (4, 6, 10,and 20 μM) of a fluorescent substrate, and varying concentrations ofCompounds II-XIII (the concentrations of Compound XII were 0, 16.2,32.5, and 54.1 nM; the concentrations of all other compounds were 0,165, 330, and 550 nM). The K_(i) values were estimated fromdouble-reciprocal plots of initial velocities as a function of substrateconcentration.

High-salt conditions comprised potassium HEPES buffer at pH 7.4 (10 mM),KCl (150 mM), LF (about 33 nM), varying concentrations (10, 20, 40, and100 μM) of a fluorescent substrate, and varying concentrations ofCompounds II-XIII (as described above for the low-salt conditions). TheK_(i) values were estimated as in the low-salt conditions. Eachexperiment was performed also in the presence of 0.1 mg/mL BSA (datapresented in parantheses). All assays were performed in triplicate andanalogous results were obtained in at least two or three experiments.TABLE 20 Apparent inhibition constants (K_(i)) of the commercialneomycin B (I) and its synthetic derivatives II-XIII for the proteaseactivity of LF at various assay conditions. K_(i) (nM) K_(i) (μM)Aminoglycosides Low salt High salt Neomycin B (I) 37 ± 2 (34 ± 0.4) 59 ±6 (64 ± 8) II 11 ± 2 50 ± 7 III 0.5 ± 0.1 (17 ± 4) 28 ± 6 (30 ± 5) IV 13± 2 66 ± 9 V 28 ± 2 134 ± 17 VI 1.3 ± 0.4 39 ± 6 VII 15 ± 2 (36 ± 5) 85± 11 (58 ± 8) VIII 0.6 ± 0.1 (15 ± 3) 20 ± 3 (24 ± 3) IX 0.2 ± 0.1 10 ±2 X 0.4 ± 0.1 21 ± 4 XI 52 ± 5 81 ± 21 XII 23 ± 2 125 ± 25 XIII 0.7 ±0.2 (33 ± 6) 1.1 ± 0.2 (1.2 ± 0.2)

As is shown in Table 20, all the tested compounds were found to becompetitive inhibitors. From the measured apparent K_(i) values at lowionic strength assay conditions (low salt), it was found that among theanalogues tested, 6 compounds (III, VI, VIII, IX, X, XIII), having K_(i)values in the range of 0.2-1.3 nM, are predominantly better inhibitorsthan the neomycin B itself (K_(i)=37 mM). It was further found that thebinding affinity of the analogues of gluco series (having a glucosesubstituent at ring V) increases gradually with increasing number ofamino groups on the ring: (2NH₂)glucose (II, VIII)>(1NH₂)glucose (III,VIII)>glucose (IV, VII). In this series of compounds, no particularinfluence on the position of the amino group(s) on the glucose ring isobserved. The ring configuration, however, has a more significanteffect: the ribosamino derivative VI binds about 10-fold tighter thantwo monoamino derivatives of glucose (IV and VII), and thediamino-D-allose derivative 1, which consists of an unusualcis-1,2-diamine substitution at ring E, binds about 20-fold weaker thanthe amino-D-glucose derivatives III and VIII. These data suggest that,although the number of amino groups on the ligand is in general criticalfor LF binding affinity, structural features of the ligand play animportant role in the proper recognition of LF.

Without wishing to be bound to a particular theory, it was hypothesizedthat since the disulfide dimer XIII has twice as many amino groups asits parent “monomeric” IX, its binding affinity to LF would be expectedto be significantly higher. The observed similar extent of inhibition ofXIII and IX was, however, very intriguing suggesting that in the case ofthe dimer XIII, in addition to a “specific” active site binding, anadditional “nonspecific interaction” with the LF protein may occur.

Various studies dealing with many different protein-polyelectrolyteinteractions (31) and the interactions of aminoglycosides with a numberof ribozymes (12, 21), support this assumption. To test thispossibility, the Compounds IX and XIII, along with neomycin B, wereevaluated in the presence of 0.1 mg/mL of BSA. Again without wishing tobe bound by theory, it was found that while the binding affinities ofboth neomycin B and Compound IX were not significantly affected, thebinding of Compound XIII was reduced 47-fold by the addition of BSA,implying a nonspecific protein-ligand association in the case ofCompound XIII which increases with increased protein concentration.

Although, to date, no direct structural data on the interaction ofaminoglycosides with LF is available, and without wishing to be bound bytheory, a preliminary investigation of the binding mechanism suggeststhat the inhibitory activity of aminoglycosides is ionic strengthdependent, supporting the possibility that the predominant interactionbetween the LF and aminoglycosides may be electrostatic in origin (35).Increasing the ionic strength from 0 to 150 mM KCl drastically shiftsthe K_(i) values of all aminoglycosides towards higher concentrations byfactors of about 1500-53,000 (Table 20). These data suggest that thecompounds of the present invention, including the parent neomycin B, canbe replaced from their LF binding site even at a relatively low ionicstrength. A possible reason for the observed different sensitivities ofdifferent aminoglycosides to changes in ionic strength may be theirdifferent numbers of amino groups and their individual pKa values. Inaddition, it is likely that the pKa values of individual ammonium groupsof neomycin B and of the dimer XIII are the same, resulting in XIIIbehaving like a “monomer” and displaying the same sensitivity asneomycin B towards ionic strength (about 1500-fold, Table 20) (38).

Without wishing to be bound by theory, the observed 53-fold higheraffinity of Compound XIII compared to that of the neomycin B, both atthe low and high salt concentrations, suggests that the presence oftwice the number of charged groups in XIII may be responsible for itsincreased affinity. Such high ionic strength conditions may also be ableto overcome the nonspecific LF-XIII association, as suggested by thesame K_(i) value observed for XI, with and without the presence of BSA.Furthermore, since 150 mM KCl best resembles the physiological ionicstrength into the mammalian cell (39), XIII can be considered as apreferred aminoglycoside inhibitor of LF at seemingly physiologicalconditions.

When the new derivatives II-XIII were tested by means of surface plasmonresonance (SPR) against immobilized 27-mer RNA construct (AS-wt) (25),binding constants in the range of 0.4-2.9 μM were determined, with noobvious dependence of K_(d) on modification type (Table 21). Several ofthese derivatives, including the dimer XIII (K_(d)=0.4 μM), displayedapparently similar extent of binding affinity to that of the parentneomycin B (K_(j)=0.3 μM), showing no apparently significantcontribution of the number of amino groups on the ligand to RNA binding;Without wishing to be bound by theory, it is suggested that, unlike thebinding affinity to LF, in which increasing number of amino groups onthe natural drug lead to increased binding, a more subtle balance ofinteractions may govern the binding affinity of these ligands to RNA.

To compare the observed RNA binding affinities to antibacterialactivity, the Compounds II-XIII were further investigated against B.anthracis (Sterne strain) (40), and the minimal inhibitoryconcentrations (MIC) were determined using a microdilution assay withneomycin B as a control (Table 21). No previous studies onaminoglycoside drugs have been performed to make such a comparisonbetween rRNA binding and antibacterial activity for B. anthracis.

Minimal inhibitory concentrations (MIC) against B. anthracis and bindingconstants (K_(d)) to 16S A site RNA were tested for the commercialneomycin B and its synthetic derivatives II-XIII. For the MICmeasurements, the concentrated stock solutions of aminoglycosides wereprepared in distilled water with known concentration. Two-fold dilutionswere used in a concentration range from 0.015 to 1024 mg/L diluted in100 μL of BHI broth and poured into wells of microtitre plates (Nunc96-well flat-bottomed microtitre plates; Nunc, Roskilde, Denmark). A 10μL volume of culture containing 10⁵ cfu/mL of B. anthracis Sterne strainwas then added. Following incubation of the plates for 18 h at 37° C. inambient air, the MICs were determined as the lowest concentration of anantibacterial agent that completely inhibited visible growth of thebacteria.

The sequence of 27-mer 16S A site RNA construct used in this study was5′ BiGGCGUCACACCUUCGGGUGAAGUCGCC 3′, and the binding assays wereperformed as previously described (11).

From the MIC values, it appears that all of the synthetic derivativespossess significant antibacterial activity against B. anthracis, some ofthem displaying activity levels comparable to that of neomycin B. Inspite of the similar binding affinities of the neomycin B and the dimerXIII to 16S A site RNA, their antimicrobial activities differed by afactor of eight, suggesting that no direct correlation between rRNAbinding and antibacterial activity can be made. While this is inagreement with earlier reported data on other aminoglycoside analogues(41), further structure-activity studies within more diverse structuresof neomycin B analogues are clearly required to better understand thisissue in detail. TABLE 21 Minimal inhibitory concentrations (MIC)against B. anthracis and binding constants (K_(d)) to 16S A site RNA forthe commercial neomycin B and its synthetic derivatives II-XIII. MICK_(d) Aminoglycosides (μg mL⁻¹) (μM) Neomycin B (I) 0.25 0.3 ± 0.1 II 82.0 ± 0.2 III 2 1.3 ± 0.3 IV 2 0.9 ± 0.1 V 1 0.7 ± 0.1 VI 2 0.7 ± 0.1VII 2 0.7 ± 0.1 VIII 1 0.6 ± 0.1 IX 8 2.9 ± 0.6 X 2 1.9 ± 0.3 XI 2 1.0 ±0.2 XII 8 1.1 ± 0.2 XIII 2 0.4 ± 0.1

EXAMPLE 9 Treatment with Compounds According to the Present Invention

The above results show that the compounds according to the presentinvention can be used for treatment of a subject suffering frominfection by an infectious microorganism. Optionally, the compounds ofthe present invention can be used to treat a subject suffering from agenetic disorder, including but not limited to, cystic fibrosis,Duchenne's muscular dystrophy, or Hurler's syndrome for example.Alternatively, the compounds of the present invention can be used totreat a subject suffering from anthrax. The method preferably includesadministering the compound of the present invention to the subjectthrough a suitable route of administration.

The compounds of the present invention are potentially useful for thetreatment of a wide spectrum of different types and/or species ofbacteria, such as gram negative and gram-positive bacteria for example.

The organisms potentially amenable to therapy with one or more of thecompounds according to the present invention include a wide variety ofGram-positive and Gram-negative organisms with a variety of growthcircumstances and requirements ranging from aerobic to anaerobic growth,including but not limited to:

(a) Gram-positive bacteria, including but not limited to, Strep.pyogenes(Group A), Strep.pneumoniae, Strep.GpB, Strep.viridans,Strep.GpD-(Enterococcus), Strep.GpC and GpG, Staph.aureus,Staph.epidermidis, Bacillus subtilis, Bacillus anthraxis, Listeriamonocytogenes, Anaerobic cocci, Clostridium spp., and Actinomyces spp;and

(b) Gram-negative bacteria, including but not limited to, Escherichiacoli, Enterobacter aerogenes, Kiebsiella pneumoniae, Proteus mirabilis,Proteus vulgaris, Morganella morganii, Providencia stuartii, Serratiamarcescens, Citrobacter freundii, Salmonella typhi, Salmonellaparatyphi, Salmonella typhi murium, Salmonella virchow, Shigella spp.,Yersinia enterocolitica, Acinetobacter calcoaceticus, Flavobacteriumspp., Haemophilus influenzae, Pseudomonas aeruginosa, Campylobacterjejuni, Vibrio parahaemolyticus, Brucella spp., Neisseria meningitidis,Neisseria gonorrhoea, Bacteroides fragilis, and Fusobacterium spp.;

(c) optionally including other organisms such as a Mycobacteria strain,including but not limited to, Mycobacterium tuberculosis, Mycobateriumsmegmatis and other Mycobacteria.

It should be noted that the term “treatment” also includes ameliorationor alleviation of a pathological condition and/or one or more symptomsthereof, curing such a condition, or preventing the genesis of such acondition.

The compounds of the present invention can be used to produce apharmaceutical composition. Thus, according to another aspect of thepresent invention there is provided a pharmaceutical composition whichincludes, as an active ingredient thereof, a compound and apharmaceutical acceptable carrier. As used herein a “pharmaceuticalcomposition” refers to a preparation of one or more of the activeingredients described herein, either compounds or physiologicallyacceptable salts thereof, with other chemical components such astraditional drugs, physiologically suitable carriers and excipients. Thepurpose of a pharmaceutical composition is to facilitate administrationof a compound or cell to an organism. Pharmaceutical compositions of thepresent invention may be manufactured by processes well known in theart, e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes.

In a preferred embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans.Hereinafter, the phrases “physiologically suitable carrier” and“pharmaceutically acceptable carrier” are interchangeably used and referto an approved carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered conjugate.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the therapeutic is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of thecompound, preferably in purified form, together with a suitable amountof carrier so as to provide the form for proper administration to thepatient. The formulation should be suitable for the mode ofadministration.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate processes andadministration of the active ingredients. Examples, without limitation,of excipients include calcium carbonate, calcium phosphate, varioussugars and types of starch, cellulose derivatives, gelatin, vegetableoils and polyethylene glycols.

Further techniques for formulation and administration of activeingredients may be found in “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., latest edition, which is incorporatedherein by reference as if fully set forth herein.

The pharmaceutical compositions herein described may also comprisesuitable solid or gel phase carriers or excipients. Examples of suchcarriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, transdermal, intestinal or parenteral delivery,including intramuscular, subcutaneous and intramedullary injections aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore pharmaceutically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological salinebuffer. For transmucosal administration, penetrants are used in theformulation. Such penetrants are generally known in the art.

For oral administration, the active ingredients can be formulatedreadily by combining the active ingredients with pharmaceuticallyacceptable carriers well known in the art. Such carriers enable theactive ingredients of the invention to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for oral ingestion by a patient. Pharmacological preparations fororal use can be made using a solid excipient, optionally grinding theresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active ingredient doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the active ingredient and a suitable powderbase such as lactose or starch.

The active ingredients described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidsesters such as ethyl oleate, triglycerides or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of theactive ingredients to allow for the preparation of highly concentratedsolutions.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, pharmaceuticalcompositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The pharmaceutical compositions of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with anions such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with cations suchas those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The active ingredients of the present invention may also be formulatedin rectal compositions such as suppositories or retention enemas, using,e.g., conventional suppository bases such as cocoa butter or otherglycerides.

The pharmaceutical compositions herein described may also comprisesuitable solid of gel phase carriers or excipients. Examples of suchcarriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

The topical route is optionally performed, and is assisted by a topicalcarrier. The topical carrier is one which is generally suited fortopical active ingredient administration and includes any such materialsknown in the art. The topical carrier is selected so as to provide thecomposition in the desired form, e.g., as a liquid or non-liquidcarrier, lotion, cream, paste, gel, powder, ointment, solvent, liquiddiluent, drops and the like, and may be comprised of a material ofeither naturally occurring or synthetic origin. It is essential,clearly, that the selected carrier does not adversely affect the activeagent or other components of the topical formulation, and which isstable with respect to all components of the topical formulation.Examples of suitable topical carriers for use herein include water,alcohols and other nontoxic organic solvents, glycerin, mineral oil,silicone, petroleum jelly, lanolin, fatty acids, vegetable oils,parabens, waxes, and the like. Preferred formulations herein arecolorless, odorless ointments, liquids, lotions, creams and gels.

Ointments are semisolid preparations, which are typically based onpetrolatum or other petroleum derivatives. The specific ointment base tobe used, as will be appreciated by those skilled in the art, is one thatwill provide for optimum active ingredients delivery, and, preferably,will provide for other desired characteristics as well, e.g., emolliencyor the like. As with other carriers or vehicles, an ointment base shouldbe inert, stable, nonirritating and nonsensitizing. As explained inRemington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.:Mack Publishing Co., 1995), at pages 1399-1404, ointment bases maybegrouped in four classes: oleaginous bases; emulsifiable bases; emulsionbases; and water-soluble bases. Oleaginous ointment bases include, forexample, vegetable oils, fats obtained from animals, and semisolidhydrocarbons obtained from petroleum. Emulsifiable ointment bases, alsoknown as absorbent ointment bases, contain little or no water andinclude, for example, hydroxystearin sulfate, anhydrous lanolin andhydrophilic petrolatum. Emulsion ointment bases are either water-in-oil(W/O) emulsions or oil-in-water (O/W) emulsions, and include, forexample, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid.Preferred water-soluble ointment bases are prepared from polyethyleneglycols of varying molecular weight; again, reference may be made toRemington: The Science and Practice of Pharmacy for further information.

Lotions are preparations to be applied to the skin surface withoutfriction, and are typically liquid or semiliquid preparations, in whichsolid particles, including the active agent, are present in a water oralcohol base. Lotions are usually suspensions of solids, and maycomprise a liquid oily emulsion of the oil-in-water type. Lotions arepreferred formulations herein for treating large body areas, because ofthe ease of applying a more fluid composition. It is generally necessarythat the insoluble matter in a lotion be finely divided. Lotions willtypically contain suspending agents to produce better dispersions aswell as active ingredients useful for localizing and holding the activeagent in contact with the skin, e.g., methylcellulose, sodiumcarboxymethylcellulose, or the like.

Creams containing the selected active ingredients are, as known in theart, viscous liquid or semisolid emulsions, either oil-in-water orwater-in-oil. Cream bases are water-washable, and contain an oil phase,an emulsifier and an aqueous phase. The oil phase, also sometimes calledthe “internal” phase, is generally comprised of petrolatum and a fattyalcohol such as cetyl or stearyl alcohol; the aqueous phase usually,although not necessarily, exceeds the oil phase in volume, and generallycontains a humectant. The emulsifier in a cream formulation, asexplained in Remington, supra, is generally a nonionic, anionic,cationic or amphoteric surfactant.

Gel formulations are preferred for application to the scalp. As will beappreciated by those working in the field of topical active ingredientsformulation, gels are semisolid, suspension-type systems. Single-phasegels contain organic macromolecules distributed substantially uniformlythroughout the carrier liquid, which is typically aqueous, but also,preferably, contain an alcohol and, optionally, an oil.

Various additives, known to those skilled in the art, may be included inthe topical formulations of the invention. For example, solvents may beused to solubilize certain active ingredients substances. Other optionaladditives include skin permeation enhancers, opacifiers, anti-oxidants,gelling agents, thickening agents, stabilizers, and the like.

The topical compositions of the present invention may also be deliveredto the skin using conventional dermal-type patches or articles, whereinthe active ingredients composition is contained within a laminatedstructure, that serves as a drug delivery device to be affixed to theskin. In such a structure, the active ingredients composition iscontained in a layer, or “reservoir”, underlying an upper backing layer.The laminated structure may contain a single reservoir, or it maycontain multiple reservoirs. In one embodiment, the reservoir comprisesa polymeric matrix of a pharmaceutically acceptable contact adhesivematerial that serves to affix the system to the skin during activeingredients delivery. Examples of suitable skin contact adhesivematerials include, but are not limited to, polyethylenes, polysiloxanes,polyisobutylenes, polyacrylates, polyurethanes, and the like. Theparticular polymeric adhesive selected will depend on the particularactive ingredients, vehicle, etc., i.e., the adhesive must be compatiblewith all components of the active ingredients-containing composition.Alternatively, the active ingredients-containing reservoir and skincontact adhesive are present as separate and distinct layers, with theadhesive underlying the reservoir which, in this case, may be either apolymeric matrix as described above, or it may be a liquid or hydrogelreservoir, or may take some other form.

The backing layer in these laminates, which serves as the upper surfaceof the device, functions as the primary structural element of thelaminated structure and provides the device with much of itsflexibility. The material selected for the backing material should beselected so that it is substantially impermeable to the activeingredients and to any other components of the activeingredients-containing composition, thus preventing loss of anycomponents through the upper surface of the device. The backing layermay be either occlusive or non-occlusive, depending on whether it isdesired that the skin become hydrated during active ingredientsdelivery. The backing is preferably made of a sheet or film of apreferably flexible elastomeric material. Examples of polymers that aresuitable for the backing layer include polyethylene, polypropylene, andpolyesters.

During storage and prior to use, the laminated structure includes arelease liner. Immediately prior to use, this layer is removed from thedevice to expose the basal surface thereof, either the activeingredients reservoir or a separate contact adhesive layer, so that thesystem may be affixed to the skin. The release liner should be made froman active ingredients/vehicle impermeable material.

Such devices may be fabricated using conventional techniques, known inthe art, for example by casting a fluid admixture of adhesive, activeingredients and vehicle onto the backing layer, followed by laminationof the release liner. Similarly, the adhesive mixture may be cast ontothe release liner, followed by lamination of the backing layer.Alternatively, the active ingredients reservoir may be prepared in theabsence of active ingredients or excipient, and then loaded by “soaking”in an active ingredients/vehicle mixture.

As with the topical formulations of the invention, the activeingredients composition contained within the active ingredientsreservoirs of these laminated system may contain a number of components.In some cases, the active ingredients may be delivered “neat,” i.e., inthe absence of additional liquid. In most cases, however, the activeingredients will be dissolved, dispersed or suspended in a suitablepharmaceutically acceptable vehicle, typically a solvent or gel. Othercomponents, which may be present, include preservatives, stabilizers,surfactants, and the like.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredient effective to prevent, alleviate or ameliorate symptomsof disease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any active ingredient used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromactivity assays in animals. For example, a dose can be formulated inanimal models to achieve a circulating concentration range that includesthe IC₅₀ as determined by activity assays.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures inexperimental animals, e.g., by determining the IC₅₀ and the LD₅₀ (lethaldose causing death in 50% of the tested animals) for a subject activeingredient. The data obtained from these activity assays and animalstudies can be used in formulating a range of dosage for use in human.For example, therapeutically effective doses suitable for treatment ofgenetic disorders can be determined from the experiments with animalmodels of these diseases.

The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain themodulating effects, termed the minimal effective concentration (MEC).The MEC will vary for each preparation, but may optionally be estimatedfrom whole animal data.

Dosage intervals can also be determined using the MEC value.Preparations may optionally be administered using a regimen, whichmaintains plasma levels above the MEC for 10-90% of the time, preferablebetween 30-90% and most preferably 50-90%.

Depending on the severity and responsiveness of the condition to betreated, dosing can also be a single administration of a slow releasecomposition described hereinabove, with course of treatment lasting fromseveral days to several weeks or until cure is effected or diminution ofthe disease state is achieved.

Suppositories generally contain active ingredient in the range of fromabout 0.5% to about 10% by weight; oral formulations preferably containfrom about 10% to about 95% active ingredient.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accompanied by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising an active ingredient of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition.

As used herein, the term “modulate” includes substantially inhibiting,slowing or reversing the progression of a disease, substantiallyameliorating clinical symptoms of a disease or condition, orsubstantially preventing the appearance of clinical symptoms of adisease or condition. A “modulator” therefore includes an agent whichmay modulate a disease or condition. Modulation of viral, protozoa andbacterial infections includes any effect which substantially interrupts,prevents or reduces any viral, bacterial or protozoa activity and/orstage of the virus, bacterium or protozoon life cycle, or which reducesor prevents infection by the virus, bacterium or protozoon in a subject,such as a human or lower animal.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent and patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

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1. A compound having the general formula I:

wherein: R₁ is a monosaccharide residue or an oligosaccharide residue; Xand Y are each independently oxygen or sulfur; R₂ and R₃ are eachindependently selected from the group consisting of hydrogen, hydroxy,thiol, amine, alkyl, cycloalkyl, aryl, alkoxy, aryloxy, thioalkoxy andthioaryloxy; and wherein the carbon at the fifth position of ring B hasan R configuration or an S configuration; or a pharmaceuticallyacceptable salt thereof.
 2. The compound of claim 1, wherein X isoxygen.
 3. The compound of claim 2, wherein Y is oxygen.
 4. The compoundof claim 3, wherein R₁ is a monosaccharide residue.
 5. The compound ofclaim 4, wherein said monosaccharide residue is a five-membered(furanose) or a six-membered (pyranose) monosaccharide residue.
 6. Thecompound of claim 4, wherein said monosaccharide residue comprises atleast one amine group and/or at least one aminoalkyl group.
 7. Thecompound of claim 6, wherein said at least one amine group and/or saidat least one aminoalkyl group is at one or more of positions 2, 3, 4 or5.
 8. The compound of claim 7, wherein said at least one aminoalkylgroup is an aminomethyl group (CH₂—NH₂).
 9. The compound of claim 8,wherein said monosaccharide residue is a pyranose monosaccharideresidue, and said aminomethyl group is at position
 5. 10. The compoundof claim 7, wherein said monosaccharide residue is a pyranosemonosaccharide residue, and said at least one amine group is at one ormore of positions 2, 3 or
 4. 11. The compound of claim 7, wherein saidmonosaccharide residue is a furanose monosaccharide residue, and saidaminoalkyl group is at position
 4. 12. The compound of claim 4, whereinsaid monosaccharide residue is a L-monosaccharide or a D-monosaccharide.13. The compound of claim 1, wherein R₁ is an oligosaccharide residue.14. The compound of claim 13, wherein said oligosaccharide residuecomprises at least two monosaccharide residues, wherein each isindependently a five-membered (furanose) or a six-membered (pyranose)monosaccharide residue.
 15. The compound of claim 14, wherein at leastone of said at least two monosaccharide residues comprises at least oneamine group and/or at least one aminoalkyl group.
 16. The compound ofclaim 15, wherein said at least one amine group is at position 2 of apyranose monosaccharide residue.
 17. The compound of claim 16, whereinsaid at least one aminoalkyl group is at position 5 of a pyranosemonosaccharide residue.
 18. The compound of claim 14, wherein saidoligosaccharide comprises a furanose monosaccharide linked to a pyranosemonosaccharide.
 19. The compound of claim 14, wherein each of said atleast two monosaccharide residues is independently a D-monosaccharide ora L-monosaccharide.
 20. The compound of claim 1, wherein X is sulfur andR₁ is a monosaccharide residue.
 21. The compound of claim 20, whereinsaid monosaccharide is a furanose monosaccharide residue.
 22. Thecompound of claim 13, wherein said oligosaccharide residue comprises atleast four monosaccharide residues, wherein each monosaccharide residueis independently a five-membered (furanose) or a six-membered (pyranose)monosaccharide residue.
 23. The compound of claim 22, wherein saidoligosaccharide residue is selected from the group consisting of aNeomycin B residue, a Paromomycin residue, a Ribostamycin residue, aGentamycin residue, a Amikacin residue, a Neamine residue, a Nebramineresidue and a Tobramine residue.
 24. A pharmaceutical formulationcomprising a therapeutically effect amount the compound of claim 1 and apharmaceutically acceptable carrier.
 25. A compound having the generalformula II:

wherein: Y is oxygen or sulfur; R₂ and R₃ are each independentlyhydrogen, hydroxy, thiol, amine, alkyl, cycloalkyl, aryl, alkoxy,aryloxy, thioalkoxy or thioaryloxy, and wherein the carbon at the fifthposition of ring B has an R configuration or an S configuration; or apharmaceutically acceptable salt thereof.
 26. The compound of claim 25,wherein Y is oxygen, and R₂ and R₃ are both hydroxy.
 27. Apharmaceutical formulation comprising a therapeutically effective amountof the compound of claim 1 and a pharmaceutically acceptable carrier.28. A compound having the general formula III:

wherein: R₁ is an oligosaccharide residue comprising at least twomonosaccharide residues, wherein each is independently a five-membered(furanose) or a six-membered (pyranose) monosaccharide residue. Y isoxygen or sulfur; X is disulfide; R₂ and R₃ are each independentlyselected from the group consisting of hydrogen, hydroxy, thiol, amine,alkyl, cycloalkyl, aryl, alkoxy, aryloxy, thioalkoxy and thioaryloxy;and wherein the carbon at the fifth position of ring B has an Rconfiguration or an S configuration; or a pharmaceutically acceptablesalt thereof.
 29. The compound of claim 28, wherein Y is oxygen.
 30. Thecompound of claim 28, wherein R₂ and R₃ are both hydroxy.
 31. Thecompound of claim 28, wherein said oligosaccharide residue comprises atleast four monosaccharide residues, each of said monosaccharide residuesis independently a five-membered (furanose) or a six-membered (pyranose)monosaccharide residue.
 32. The compound of claim 31, wherein saidoligosaccharide residue is selected from the group consisting of aParomomycin residue, a Ribostamycin residue, a Gentamycin residue, aAmikacin residue, a Neamine residue, a Nebramine residue and a Tobramineresidue.
 33. A pharmaceutical formulation comprising a therapeuticallyeffective amount of the compound of claim 28 and a pharmaceuticallyacceptable carrier.
 34. A method for treating a subject in need thereof,comprising: administering to the subject a therapeutically effectiveamount of the compound of claim
 1. 35. A method for treating a subjecthaving an infectious micro-organism, comprising: administering to thesubject a therapeutically effective amount of the compound of claim 1.36. The method of claim 35, wherein said infectious microorganismcomprises a bacterial strain.
 37. The method of claim 36, wherein saidbacterial strain is resistant to at least one antibiotic.
 38. The methodof claim 36, wherein said bacterial strain comprises one or more ofGram-positive and Gram-negative organisms with a variety of growthcircumstances and requirements ranging from aerobic to anaerobic growth.39. The method of claim 38, wherein said bacterial strain is selectedfrom the group consisting of: (a) Gram-positive bacteria selected fromthe group consisting of Strep.pyogenes (Group A), Strep.pneumoniae,Strep.GpB, Strep.viridans, Strep.GpD-(Enterococcus), Strep.GpC and GpG,Staph.aureus, Staph.epidermidis, Bacillus subtilis, Bacillus anthraxis,Listeria monocytogenes, Anaerobic cocci, Clostridium spp., andActinomyces spp; and (b) Gram-negative bacteria selected from the groupconsisting of Escherichia coli, Enterobacter aerogenes, Kiebsiellapneumoniae, Proteus mirabilis, Proteus vulgaris, Morganella morganii,Providencia stuartii, Serratia marcescens, Citrobacter freundii,Salmonella typhi, Salmonella paratyphi, Salmonella typhi murium,Salmonella virchow, Shigella spp., Yersinia enterocolitica,Acinetobacter calcoaceticus, Flavobacterium spp., Haemophilusinfluenzae, Pseudomonas aueroginosa, Campylobacter jejuni, Vibrioparahaemolyticus, Brucella spp., Neisseria meningitidis, Neisseriagonorrhoea, Bacteroides fragilis, and Fusobacterium spp.
 40. The methodof claim 36, wherein said bacterial strain comprises a Mycobacteriastrain.
 41. The method of claim 40, wherein said Mycobacteria strain isselected from the group consisting of Mycobacterium tuberculosis orMycobaterium smegmatis.
 42. The method of claim 39 wherein saidbacterial strain comprises Bacillus anthraxis.
 43. A method for treatinga subject having a genetic disorder, comprising: administering atherapeutically effective amount of the compound of claim
 1. 44. Themethod of claim 43, wherein the genetic disorder comprises a proteinhaving a truncation mutation.
 45. The method of claim 44, wherein thegenetic disorder comprises cystic fibrosis.
 46. The method of claim 44,wherein the genetic disorder comprises Duchenne's muscular dystrophy orHurler's syndrome.
 47. A method for treating anthrax, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the compound of claim
 1. 48. A method for treating a subjectin need thereof, comprising: administering to the subject atherapeutically effective amount of the compound of claim
 25. 49. Amethod for treating a subject having an infectious micro-organism,comprising: administering to the subject a therapeutically effectiveamount of the compound of claim
 25. 50. The method of claim 49, whereinsaid infectious microorganism comprises a bacterial strain.
 51. Themethod of claim 50, wherein said bacterial strain is resistant to atleast one antibiotic.
 52. The method of claim 50, wherein said bacterialstrain comprises one or more of Gram-positive and Gram-negativeorganisms with a variety of growth circumstances and requirementsranging from aerobic to anaerobic growth.
 53. The method of claim 52,wherein said bacterial strain is selected from the group consisting of:(a) Gram-positive bacteria selected from the group consisting ofStrep.pyogenes (Group A), Strep.pneumoniae, Strep.GpB, Strep.viridans,Strep.GpD-(Enterococcus), Strep.GpC and GpG, Staph.aureus,Staph.epidermidis, Bacillus subtilis, Bacillus anthraxis, Listeriamonocytogenes, Anaerobic cocci, Clostridium spp., and Actinomyces spp;and (b) Gram-negative bacteria selected from the group consisting ofEscherichia coli, Enterobacter aerogenes, Kiebsiella pneumoniae, Proteusmirabilis, Proteus vulgaris, Morganella morganii, Providencia stuartii,Serratia marcescens, Citrobacter freundii, Salmonella typhi, Salmonellaparatyphi, Salmonella typhi murium, Salmonella virchow, Shigella spp.,Yersinia enterocolitica, Acinetobacter calcoaceticus, Flavobacteriumspp., Haemophilus influenzae, Pseudomonas aueroginosa, Campylobacterjejuni, Vibrio parahaemolyticus, Brucella spp., Neisseria meningitidis,Neisseria gonorrhoea, Bacteroides fragilis, and Fusobacterium spp. 54.The method of claim 50, wherein said bacterial strain comprises aMycobacteria strain.
 55. The method of claim 54, wherein saidMycobacteria strain is selected from the group consisting ofMycobacterium tuberculosis or Mycobaterium smegmatis.
 56. The method ofclaim 54 wherein said bacterial strain comprises Bacillus anthraxis. 57.A method for treating a subject having a genetic disorder, comprising:administering a therapeutically effective amount of the compound ofclaim
 25. 58. The method of claim 57, wherein the genetic disordercomprises a protein having a truncation mutation.
 59. The method ofclaim 57, wherein the genetic disorder comprises cystic fibrosis. 60.The method of claim 57, wherein the genetic disorder comprisesDuchenne's muscular dystrophy or Hurler's syndrome.
 61. A method fortreating anthrax, comprising administering to a subject in need thereofa therapeutically effective amount of the compound of claim
 25. 62. Amethod for treating a subject in need thereof, comprising: administeringto the subject a therapeutically effective amount of the compound ofclaim
 28. 63. A method for treating a subject having an infectiousmicro-organism, comprising: administering to the subject atherapeutically effective amount of the compound of claim
 28. 64. Themethod of claim 63, wherein said infectious microorganism comprises abacterial strain.
 65. The method of claim 64, wherein said bacterialstrain is resistant to at least one antibiotic.
 66. The method of claim64, wherein said bacterial strain comprises one or more of Gram-positiveand Gram-negative organisms with a variety of growth circumstances andrequirements ranging from aerobic to anaerobic growth.
 67. The method ofclaim 64, wherein said bacterial strain is selected from the groupconsisting of: (a) Gram-positive bacteria selected from the groupconsisting of Strep.pyogenes (Group A), Strep.pneumoniae, Strep.GpB,Strep.viridans, Strep.GpD-(Enterococcus), Strep.GpC and GpG,Staph.aureus, Staph.epidermidis, Bacillus subtilis, Bacillus anthraxis,Listeria monocytogenes, Anaerobic cocci, Clostridium spp., andActinomyces spp; and (b) Gram-negative bacteria selected from the groupconsisting of Escherichia coli, Enterobacter aerogenes, Kiebsiellapneumoniae, Proteus mirabilis, Proteus vulgaris, Morganella morganii,Providencia stuartii, Serratia marcescens, Citrobacter freundii,Salmonella typhi, Salmonella paratyphi, Salmonella typhi murium,Salmonella virchow, Shigella spp., Yersinia enterocolitica,Acinetobacter calcoaceticus, Flavobacterium spp., Haemophilusinfluenzae, Pseudomonas aueroginosa, Campylobacter jejuni, Vibrioparahaemolyticus, Brucella spp., Neisseria meningitidis, Neisseriagonorrhoea, Bacteroides fragilis, and Fusobacterium spp.
 68. The methodof claim 64, wherein said bacterial strain comprises a Mycobacteriastrain.
 69. The method of claim 68, wherein said Mycobacteria strain isselected from the group consisting of Mycobacterium tuberculosis orMycobaterium smegmatis.
 70. The method of claim 67, wherein saidbacterial strain comprises Bacillus anthraxis.
 71. A method for treatinga subject having a genetic disorder, comprising: administering atherapeutically effective amount of the compound of claim
 28. 72. Themethod of claim 71, wherein the genetic disorder comprises a proteinhaving a truncation mutation.
 73. The method of claim 71, wherein thegenetic disorder comprises cystic fibrosis.
 74. The method of claim 71,wherein the genetic disorder comprises Duchenne's muscular dystrophy orHurler's syndrome.
 75. A method for treating anthrax, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the compound of claim
 28. 76. Ethyl2-O-Benzoyl-3,4-dideoxy-3,4-diazido-6-O-chloroacetyl-1-thio-β-D-allopyranoside(compound 9). 77.p-Methylphenyl-4,6-dideoxy-4,6-diazido-2,3-O-benzoyl-1-thio-β-D-glucopyranoside(compound 5 f).
 78. Ethyl3,4-di-O-benzoyl-6-O-chloroacetyl-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranose(compound 10).
 79. Ethyl 2,3,5-Tri-O-acetyl-1-thio-D-ribofuranose(compound 11). 80.p-Methylphenyl-2-deoxy-2-phthalimido-6-deoxy-6-azido-3,4-di-O-benzoyl-1-thio-β-D-glucopyranoside(compound 5 e). 81.p-Methylphenyl-6-O-Acetyl-4-deoxy-4-azido-2,3-di-O-benzoyl-1-thio-β-D-glucopyranoside(compound 5 c). 82.p-Methylphenyl-5-deoxy-5-azido-2,3-di-O-benzoyl-1-thio-D-ribofuranose(compound 8 b). 83.p-Methylphenyl-5-deoxy-5-O-benzoyl-2,3-diazido-1-thio-D-ribofuranose(compound 24). 84.p-methylphenyl-2-O-Benzoyl-3,4-dideoxy-3,4-diazido-6-O-chloroacetyl-1-thio-β-D-allopyranoside(compound 7 a).
 85. p-Methylphenyl3,4-di-O-acetyl-2,6-dideoxy-2,6-diazido-β-L-idopyranoside(1-3)-2-O-acetyl-5-O-tert-butyldiphenylsylil-1-thio-β-D-ribofuranoside(Compound 19 b).
 86. A compound having the general formula IV:

wherein: each of Z₁ and Z₂ is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aryl, a hydroxy protectinggroup, an amino protecting group and a thiol protecting group. each ofT₁-T₄ is independently a hydroxy protecting group; each of Q₁-Q₆ isindependently an amino protecting group; X is oxygen or sulfur; Y isoxygen or sulfur; and wherein the carbon at the fifth position of ring Bhas an R configuration or an S configuration.
 87. The compound of claim86, wherein each of said hydroxy protecting groups is an O-acetyl group(OT₁-OT₄).
 88. The compound of claim 86, wherein each of said aminoprotecting groups is an azido group.
 89. The compound of claim 86,wherein X is oxygen, each of said Z₁, Z₂ and OT₁-OT₄ is an O-acetylgroup and each of said NQ₁-NQ₆ is an azido group.
 90. The compound ofclaim 86, wherein X is oxygen, each of said Z₁, Z₂ is hydrogen, each ofsaid OT₁-OT₄ is an O-acetyl group and each of said NQ₁-NQ₆ is an azidogroup.
 91. The compound of claim 86, wherein X is sulfur, each of saidZ₁, Z₂ and OT₁-OT₄ is an O-acetyl group and each of said NQ₁-NQ₆ is anazido group.
 92. A method of synthesizing the compound of claim 1, themethod comprising: (a) providing a compound having the general formulaIV:

wherein: each of Z₁ and Z₂ is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aryl, a hydroxy protectinggroup, an amino protecting group and a thiol protecting group. each ofT₁-T₄ is independently a hydroxy protecting group; each of Q₁-Q₆ isindependently an amino protecting group; X is oxygen or sulfur; Y isoxygen or sulfur; and wherein the carbon at the fifth position of ring Bhas an R configuration or an S configuration; (b) providing a compoundhaving the general formula V, VI or VII:

wherein: each of G, I, J, K, U and V is independently selected from thegroup consisting of a hydroxy protecting group and an amino protectinggroup; SL is a thiolated leaving group; and each of the carbons atpositions 1, 3 and 4 in Formula I and at position 1 in Formula II has anR configuration or an S configuration; (c) coupling said compound havingsaid general formula IV and said compound having said general formula I,II or IV; and (d) removing each of said hydroxy protecting groups andsaid amino protecting groups, to thereby provide the compound ofclaim
 1. 93. The method of claim 92, wherein said hydroxy protectinggroup is selected from the group consisting of O-acetyl, O-chloroacetyland O-benzoyl.
 94. The method of claim 92, wherein said amino protectinggroup is selected from the group consisting of an azido group and aN-phtalimido group.
 95. The method of claim 92, wherein said thiolatedleaving group is selected from the group consisting of thioethyl andpara-thiotoluene.
 96. A method of synthesizing the compound of claim 25,the method comprising: (a) providing a compound having the generalformula IV:

wherein: each of Z₁ and Z₂ is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aryl, a hydroxy protectinggroup, an amino protecting group and a thiol protecting group. each ofT₁-T₄ is independently a hydroxy protecting group; each of Q₁-Q₆ isindependently an amino protecting group; X is sulfur; Y is oxygen orsulfur; and wherein the carbon at the fifth position of ring B has an Rconfiguration or an S configuration; and (b) removing each of saidhydroxy protecting groups and said amino protecting groups, to therebyprovide the compound of claim
 25. 97. The method of claim 96, whereinsaid hydroxy protecting group is selected from the group consisting ofO-acetyl, O-chloroacetyl and O-benzoyl.
 98. The method of claim 96,wherein said amino protecting group, is selected from the groupconsisting of an azido group and a N-phtalimido group.
 99. A method ofsynthesizing the compound of claim 28, the method comprising: (a)providing a compound having the general formula IV:

wherein: each of Z₁ and Z₂ is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aryl, a hydroxy protectinggroup, an amino protecting group and a thiol protecting group; each ofT₁-T₄ is independently a hydroxy protecting group; each of Q₁-Q₆ isindependently an amino protecting group; X is sulfur; Y is oxygen orsulfur; and wherein the carbon at the fifth position of ring B has an Rconfiguration or an S configuration; (b) providing an oligosaccharidecomprised of at least four monosaccharide residues and having at leastone free thiol group attached to at least one of said monosaccharideresidues, wherein any hydroxy group or amino group attached to saidmonosaccharide resiudes is protected by a hydroxy protecting group or anamino protecting group, respectively; (c) coupling said compound havingsaid general formula IV and said oligosaccharide, to thereby form adisulfide bond therebetween; and (d) removing each of said hydroxyprotecting groups and said amino protecting groups, to thereby providethe compound of claim
 28. 100. The method of claim 99, wherein saidoligosaccharide has said general formula IV.
 101. The method of claim99, wherein said hydroxy protecting group is selected from the groupconsisting of O-acetyl, O-chloroacetyl and O-benzoyl.
 102. The method ofclaim 99, wherein said amino protecting group is selected from the groupconsisting of an azido group and a N-phtalimido group.